Methods, agents and compositions for treatment of inflammatory conditions

ABSTRACT

The present invention relates to the screening, diagnosis, prognostic evaluation, and treatment or prevention of age associated inflammation, chronic inflammation, and inflammatory diseases. In particular, the present invention relates to treating or preventing inflammatory diseases (e.g. rheumatoid arthritis or spondyloarthritis) or patients with inflammatory peripheral GnRH with GnRH antagonists or drugs that lower the effects of GnRH.

FIELD OF THE INVENTION

The present invention relates to the screening, diagnosis, prognosticevaluation, and treatment or prevention of age-related inflammation,chronic inflammation, and inflammatory diseases. In particular, thepresent invention relates to methods, agents and compositions fortreating inflammatory diseases (e.g. rheumatoid arthritis orspondyloarthritis) or patients with inflammatory peripheralgonadotropin-releasing hormone (GnRH) with GnRH antagonists, includingdrugs that lower the effects of GnRH or GnRH inhibitors. Mostparticularly, the present invention relates to methods for treatinginflammatory diseases, such as rheumatoid arthritis, with the GnRHantagonist ASP1707.

BACKGROUND OF THE INVENTION

Ageing is among the largest known risk factors for human diseases.Roughly 100,000 people die each day of age-related causes. Between 2000and 2050, the proportion of the number of people aged 60 years and overis expected to increase from 605 million to 2 billion.

Chronic inflammation is associated with normal and pathological ageing.Systemic chronic inflammation can accelerate ageing (Jurk, D. et al.,Nat. Comm., 2014; doi:10.1038/ncomms5172). Many age-related diseases andageing itself are closely associated with low-level chronic inflammation(Chung H Y, et al. 2009, Ageing Res. Rev. 8 (1), 18-30). Inflammatorymarkers are significant predictors of mortality in older humans. Thisproinflammatory status of the elderly underlies biological mechanismsresponsible for the decline of physical function decline and age-relateddiseases such as Alzheimer's disease and atherosclerosis that areinitiated or worsened by systemic inflammation. Understanding of theageing process should have a prominent role in new strategies forextending the health of the older population.

Inflammatory diseases themselves accelerate the ageing process due tosystemic chronic inflammation. Many of these diseases, such asrheumatoid arthritis and multiple sclerosis accelerate cardiovasculardisease and osteoporosis, both of which are examples of age-relatedconditions. Indeed, inflammatory diseases have on average a 10 yearpremature mortality largely due to increased cardiovascular disease.

Rheumatoid arthritis is a chronic, systemic inflammatory disorder thatmay affect many tissues and organs, but principally attacks the jointsproducing an inflammatory synovitis that progresses to cartilage andbone destruction. Rheumatoid arthritis can also produce diffuseinflammation in the lungs, pericardium, pleura and sclera, and alsonodular lesions, most common in subcutaneous tissue under the skin.Although the cause of rheumatoid arthritis is unknown, autoimmunityplays a role in its chronicity and progression.

About 1% of the world's population is afflicted by rheumatoid arthritis,women three times more often than men. Onset is most frequent betweenthe ages of 40 and 50, but people of any age can be affected. It can bea disabling and painful condition which can lead to substantial loss offunctioning and mobility. It is diagnosed chiefly on symptoms and signs,but also with blood tests (e.g. a test for anti-cyclic citrullinatedpeptide (CCP) antibodies) and X-rays. Diagnosis and long-term managementare typically performed by a rheumatologist, an expert in the diseasesof joints and connective tissues.

There is no cure for ageing, chronic inflammation or inflammatorydiseases. We have improved life expectancy due to various factors,including the prevention and treatment of cardiovascular disease.Nowadays, new biologic drugs for inflammatory diseases are available.The efficacy of such drugs is still inadequate in a large proportion ofpatients. For example, only about two thirds of patients with rheumatoidarthritis respond at any level to currently available biologic drugs. Ofthese patients, a significant proportion will have to cease thesetreatments either due to loss of efficacy over time, or serious sideeffects. So the average time rheumatoid arthritis patients can use abiologic is about 2 years. Current treatments for rheumatoid arthritisinclude: corticosteroids, methotrexate, tumour necrosis factorinhibitors such as etanercept (Embrel®), adalimubab (Humira®), andinfliximab (Remicade®), and other immunomodulatory and cytotoxic agents.Whilst these treatments can be effective, many require close supervisionbecause of hazardous side-effects. Response to treatment with theseagents is variable and some patients will experience pain and jointdegeneration. Thus there is a need for additional treatments forrheumatoid arthritis and related diseases. Other inflammatory diseaseshave no disease modifying therapies available, such as progressivemultiple sclerosis.

Therefore there is a need for earlier diagnosis, improved screening andprognostic evaluation, as well as better treatment and prevention ofinflammatory diseases, chronic inflammation, and age-relatedinflammation.

SUMMARY OF THE INVENTION

The present invention relates to the screening, diagnosis, prognosticevaluation, and treatment or prevention of inflammatory conditionsincluding age-related inflammation, chronic inflammation, andinflammatory diseases. In particular, the present invention relates tomethods of treating inflammatory diseases (e.g. rheumatoid arthritis orspondyloarthritis) or patients with inflammatory peripheral GnRH withGnRH antagonists or drugs that lower the effects of GnRH, in particularthe GnRH antagonist ASP1707. Inflammatory conditions generally requireor benefit from long term treatment.

Accordingly, a first aspect of the invention provides a GnRH antagonist,preferably ASP1707, for use in the treatment or prevention of aninflammatory condition in a subject, selected from an inflammatorydisease, chronic inflammation, age-related inflammation or inflammatoryperipheral GnRH, wherein said GnRH antagonist is for long-termadministration to said subject for a period of at least 12 weeks.

Also provided according to the invention is a pharmaceutical compositioncomprising a GnRH antagonist, preferably ASP1707, for such use, togetherwith at least one pharmaceutically acceptable carrier or excipient.Thus, this aspect of the invention provides a pharmaceutical compositioncomprising a GnRH antagonist, preferably ASP1707, for use in thetreatment or prevention of an inflammatory condition in a subject,selected from an inflammatory disease, chronic inflammation, age-relatedinflammation or inflammatory peripheral GnRH, wherein said compositionis for long-term administration to said subject for a period of at least12 weeks.

In a further aspect, the invention provides use of a GnRH antagonist,preferably ASP1707, for the manufacture of a medicament for use in thetreatment or prevention of an inflammatory condition in a subject,selected from an inflammatory disease, chronic inflammation, age-relatedinflammation or inflammatory peripheral GnRH, wherein said GnRHantagonist is for long-term administration to said subject for a periodof at least 12 weeks.

In a still further aspect, the invention also provides a method oftreating or preventing an inflammatory condition in a subject, selectedfrom an inflammatory disease, chronic inflammation, age-relatedinflammation or inflammatory peripheral GnRH, said method comprisingadministering a GnRH antagonist, preferably ASP1707, to said subject,wherein said GnRH antagonist is administered long-term to said subjectfor a period of at least 12 weeks.

Embodiments of the present invention provide methods, and uses basedthereon, for the screening, diagnosis, prognostic evaluation, andtreatment or prevention of age associated inflammation, chronicinflammation, inflammatory peripheral GnRH and inflammatory diseases,comprising administering a GnRH antagonist, preferably ASP1707, to thesubject, as well as agents and compositions for such treatment. Thepresent invention is not limited to a particular inflammatory disease.Examples include, but are not limited to, rheumatoid arthritis, multiplesclerosis, systemic lupus erythematosus, ankylosing spondylitis,spondyloarthritis, psoriasis, systemic sclerosis (scleroderma),inflammatory bowel disease, multiple sclerosis, osteoarthritis and otherforms of arthritis (e.g. psoriatic arthritis) and nephritis. In oneembodiment the inflammatory conditions do not include systemic lupuserythematosus.

Patients with peripheral GnRH include, but are not limited to, thosewith age associated inflammation, chronic inflammation, autoimmunedisease, cardiovascular disease, osteoporosis, Alzheimer's disease,cataracts, cancers, cancer associated inflammation, postpartum andgonadal failure (including natural and surgical menopause, polycysticovarian syndrome and Turner's syndrome). As will be described in moredetail below, subjects with peripheral GnRH do not necessarily have aninflammatory disease and may be healthy. “Peripheral GnRH” and moreparticularly “inflammatory peripheral GnRH” are defined further below.

The present invention is not limited to a particular GnRH antagonist.Examples include, but are not limited to, cetrorelix, ganirelix,abarelix, degarelix, detirelix, iturelix, ozarelix, prazarelix,ramorelix, elagolix, relugolix, ASP1707, teverelix, D17DT GnRHvaccination, or spiroindoline derivatives as gonadotropin-releasinghormone receptor antagonists. In some embodiments, the GnRH antagonistis ASP1707.

In some embodiments, the GnRH antagonist is administered in one or morerepeated doses (e.g. several times daily, daily, weekly, monthly, orother interval) for a period of time. According to the invention as setout above the GnRH antagonist is administered for a period of at least12 weeks, (e.g. at least three months, at least 6 months, at least 1year, at least 3 years, at least 5 years, or at least 10 years, orlonger). However, as discussed below, in certain further aspects of theinvention the GnRH antagonist may be administered for a shorter period(e.g. at least one week, at least two weeks, at least one month, atleast two months). In some embodiments, the GnRH antagonist isadministered at a dose of 0.1 mg to 1000 mg (e.g. 0.1, 0.25, 0.5, 1.0,5.0, 10, 25, 50, 100, 200, 300, 400, 500, 750, or 1000 mg). In someembodiments, the GnRH antagonist is administered multiple times per day,daily, weekly, or monthly. In some embodiments, the GnRH antagonist isadministered with an initial loading dose between 20 mg to 1000 mg (e.g.20, 30, 40, 50, 60, 100, 200, 300, 400, 500, or 1000 mg) followed by alower maintenance dose administered multiple times per day, daily,weekly, or monthly, or at least every 2-12 months.

In some embodiments, a long acting GnRH antagonist such as degarelix,ozarelix or abarelix is administered weekly, or every 2-4 weekintervals, or monthly intervals or every 2-6 month intervals, or yearly,10 mg to 1000 mg, in some embodiments, with an initial loading dose ofthe long acting GnRH antagonist between 20 mg to 1000 mg. Further dosesand dosage regimes are discussed below.

In some embodiments, short acting GnRH antagonists, such as cetrorelixor ganirelix, are administered up to 6 times daily, e.g. 2, 3 or 4 to 6times daily, or daily or 2-6 times weekly or weekly or every 2-4 weeksat a dose of 0.1 mg to 30 mg, for example 0.75 mg to 30 mg or 2 mg to 30mg (e.g., 0.1, 0.25, 0.5, 1.0, 5.0, 10.0, 25, 30 mg).

In some embodiments, oral GnRH antagonists such as non-peptide oral GnRHantagonists, spiroindoline derivatives as gonadotropin-releasing hormonereceptor antagonists, relugolix, elagolix, or ASP1707 are administeredup to 6 times daily, e.g. 2, 3 or 4 to 6 times daily, or daily, or 2-6times weekly or weekly at a dose of 0.1 mg to 3 g (e.g. 0.1, 0.25, 0.5,1.0, 5.0, 10.0, 25, 50, 100, 500, 1000, 2000 or 3000 mg).

In some embodiments, the present invention combats inflammatory orage-associated bio-markers such as TNFα, IL-1, IL-6 or IGF-1, an amountdependent on their age and sex in the form of a medicament.

Certain embodiments provide for the treatment or prevention ofosteoporosis and/or increasing bone mineral density in a subject, byadministering a GnRH antagonist to the subject, or administering a GnRHantagonist, with titrated oestrogen or testosterone to baseline orhigher levels, or with an osteoporosis drug.

Some embodiments provide for treating or preventing age associatedinflammation, chronic inflammation or cardiovascular disease, ordecreasing the risk of cardiovascular disease, or decreasing a patient'srisk for developing coronary heart disease or having a cardiovascularevent, including a recurrent cardiovascular event, e.g. by decreasingHbA1c or fasting blood glucose levels, decreasing blood pressure,decreasing chronic inflammation, or increasing HDL levels or decreasingLDL levels in a subject, by administering a GnRH antagonist to thesubject, or administering a GnRH antagonist, with titrated oestrogen, ortestosterone to baseline or higher levels, or with a drug to treatcardiovascular disease.

The present invention further provides for the use of a GnRH antagonist,particularly ASP1707, in the treatment of an autoimmune disease in asubject in need thereof.

Some embodiments provide for decreasing a patient's risk for developingmetabolic syndrome or developing type II diabetes, including decreasingHbA1c or fasting glucose in a subject by administering a GnRH antagonistto the subject.

Additional embodiments provide for treating systemic sclerosis(scleroderma) in a subject, by administering a GnRH antagonist to thesubject.

Certain embodiments provide for treating multiple sclerosis in asubject, by administering a GnRH antagonist to the subject.

Additional embodiments provide for treating inflammatory bowel diseasein a subject, by administering a GnRH antagonist to the subject.

Further embodiments provide for treating psoriasis in a subject, byadministering a GnRH antagonist to the subject.

In some embodiments, the present invention provides for the use of aGnRH antagonist in the treatment of ankylosing spondylitis.

In some embodiments, the present invention provides for the use of aGnRH antagonist in the treatment of spondyloarthritis.

In some embodiments, the present invention provides the use of a GnRHantagonist in the treatment of nephritis.

In some embodiments, the present invention provides the use of a GnRHantagonist in the treatment of cancer inflammation.

In other embodiments, the GnRH antagonist may be used for decreasingHbA1c, decreasing blood pressure, or increasing HDL levels in a subject.

In other embodiments, the present invention provides methods and uses ofa GnRH antagonist to lower the levels of cytokines and/or chemokines ina subject. In some embodiments the proinflammatory cytokines are TNFα,IFNγ, IL-1β and/or IL-2. In some embodiments, the present inventionprovides the use of a GnRH antagonist to lower the levels of acute phaseproteins such as C-reactive protein (CRP) or high sensitivity CRP or thelevels of auto-antibodies such as anti-CCP antibodies.

Further embodiments of the present invention provide methods and uses oftreating an autoimmune or inflammatory disease, comprising: a)identifying subjects that exhibit one or more of: are negative foranti-CCP antibodies; are non-responders to anti-TNF therapy ordisease-modifying anti-rheumatic drugs (DMARDs); and b) administering aGnRH antagonist, particularly ASP1707, to the subjects.

In some embodiments, the present invention provides for the use ofcombination therapy, particularly titrated oestrogen or testosteronetherapy to baseline or higher levels, or a disease modifying drug, orstable or tapered prednisolone, or local topical treatment, or abiologic drug, with a GnRH antagonist in the prevention or treatment ofan inflammatory condition selected from age associated inflammation,chronic inflammation, inflammatory peripheral GnRH and inflammatorydiseases.

The present invention further provides an agent, e.g. a GnRH antagonist,particularly a conjugate comprising a GnRH antagonist linked to apolymer, and pharmaceutical compositions comprising said agent, asdescribed further herein. The present invention also provides for theuse of said agents and compositions in therapy, particularly in the usesand methods described herein.

Additional embodiments will be apparent to persons skilled in therelevant art based on the teachings contained herein.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C show images of arthritis in the foot of patient 1before and during treatment with degarelix.

FIGS. 2A-2F show ultrasound pictures of patient 5. FIG. 2A shows whitearea highlighted by arrow, which is power Doppler denoting inflammatoryactivity in shoulder. FIG. 2B shows no power Doppler in same shoulder.FIG. 2C shows white area highlighted by arrow, which is power Dopplerdenoting inflammatory activity in right MCP 4 (finger joint). FIG. 2Dshows no power Doppler in right MCP4 joint. FIG. 2E shows white areahighlighted by arrow, which is power Doppler denoting inflammatoryactivity in left MCP 4 (finger joint). FIG. 2F shows no power Doppler inleft MCP 4, and decreased joint fluid (oval).

FIGS. 3A and 3B show patient 6 before (FIG. 3A) and after (FIG. 3B)degarelix treatment.

FIG. 4 shows ultrasounds of patient 6. The first ultrasound (left) showsa large effusion (swelling indicated by black area) over the rightwrist. The second ultrasound (right) shows the same area by 3.5 weeks ofdegarelix treatment. The effusion on the right is much smaller, and nolonger painful.

FIG. 5 shows disease activity variables of patient 2 whilst beingtreated with degarelix.

FIGS. 6A-6D show foot ulcers in a lupus patient. FIGS. 6A & 6C: prior todegarelix treatment; FIGS. 6B & 6D: after degarelix treatment.

FIGS. 7A-7B show hand ulcers in a lupus patient. FIG. 7A: beforedegarelix treatment; FIG. 7B: after degarelix treatment.

FIG. 8 shows photographs of foot erythema in a patient with lupus after8 weeks of treatment with degarelix.

FIG. 9 shows hip bone mineral density in patient 1.

FIG. 10 shows hip bone mineral density in patient 2.

FIG. 11 shows a photograph of a baseline of a patient with systemicsclerosis prior to treatment. Five digital ulcers/pitting scars onfinger pulpa and a fingertip pain score 100 mm (0-100 mm) were observed.

FIG. 12 shows a photograph of the same patient as FIG. 11 after 6 weeksof treatment. The fingers are almost cleared of digital ulcers/pittingscars on finger pulpa and the fingertip pain score is 30 (0-100 mm).

FIG. 13 shows the synergistic effect of methotrexate and GnRH antagonistin rheumatoid arthritis.

FIG. 14 shows a graph depicting the association of GnRH and TNFα in theperiphery of patients with rheumatoid arthritis.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the term “GnRH antagonist” refers to an agent or drugthat decreases, blocks, inhibits, abrogates, or interferes with GnRHactivity in vivo. For example, GnRH antagonists prevent or inhibit GnRHsynthesis, and/or GnRH release, and/or GnRH function or activity. Theymay inhibit the action of GnRH by inhibiting binding of GnRH at itsreceptor, and may act at the GnRH receptor or at the GnRH molecule. Theterm “GnRH antagonist” thus includes compounds such as GnRH inhibitors,GnRH vaccinations such as GnRH-DT vaccinations consisting of the GnRHdecapeptide linked to diphtheria toxoid, GnRH receptor antagonists, e.g.selective immune cell (e.g. T cell, B cell or macrophage cell) GnRHreceptor antagonists, anti-GnRH antibodies, e.g. monoclonal antibodiesagainst GnRH, circulating GnRH receptor fusion proteins, spiroindolinederivatives as gonadotropin-releasing hormone receptor antagonists,non-peptide oral GnRH antagonists, as well as agents which act toinhibit GnRH production and/or action by other mechanisms, for exampleby downregulating GnRH production due to negative feedback mechanisms,e.g. kisspeptin antagonists, oestrogen compounds, testosteronecompounds, luteinizing hormone (LH) compounds or follicle-stimulatinghormone (FSH) compounds, hypothalamic hormones or neuropeptides. Anoestrogen, testosterone, LH or FSH compound may be any compound ormolecule or preparation which has oestrogen, testosterone, LH or FSHactivity, including in particular oestrogen, testosterone, LH or FSH, orany preparation containing a said hormone, or a derivative of any saidhormone. Examples of GnRH antagonists which may be used according to theinvention are discussed further below. In one particular embodiment theGnRH antagonist acts to inhibit GnRH activity, e.g. by acting on or atthe GnRH receptor or on GnRH itself (e.g. by binding to the receptor orGnRH). In some embodiments, a suitable GnRH antagonist prevents orinhibits GnRH receptor signaling. In some embodiments, a suitable ‘GnRHantagonist’ may be too large to cross the blood brain barrier. Suchantagonist may take the form of a conjugate of a GnRH antagonist with apolymeric partner, e.g. a polymer such as a polypeptide (e.g. a proteinsuch as albumin), polysaccharide, or other polymer, such as polyethyleneglycol (PEG) e.g. pegylated GnRH inhibitors, or GnRH inhibitors fused toproteins such as albumin. A polymer is defined broadly herein to includeany compound having a multiplicity of repeating monomer units orresidues and includes oligomers. A “multiplicity” may be 2 or more, e.g.3, 4, 5, or 6 or more, but typically will be higher, e.g. 10, 12, 15,18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80 or more. A polypeptide mayaccordingly include longer polypeptide sequences such as proteins aswell as shorter peptides. Such conjugates of a GnRH antagonist with apolymer, and pharmaceutical compositions containing them, represent anovel aspect of this invention and are discussed further below.

Conjugates of a drug molecule/active agent with a polymer molecule arewidely used and reported for drug delivery, as indeed is the use ofpolymers in formulation of drug delivery systems. The polymer may bewater-soluble. The physical and chemical properties of the polymerstypically used in polymer-drug conjugates are specially synthesized toflow through the kidneys and liver without getting filtered out,allowing the drugs to be used more effectively. Further, the polymer maybe degraded e.g. through enzymes and acidity. Polymers may besynthesized to be sensitive to specific enzymes that are very apparentwith diseased tissue. The drugs remain attached to the polymer and arenot activated until the enzymes associated with the diseased tissue arepresent. This process significantly minimizes damage to healthy tissue.Any such polymer as is typically used in such drug delivery formulationsor conjugates may be used to prepare a conjugate of a polymer with aGnRH antagonist according to the present invention. Examples include butare not limited to poly(ethylene glycol) (PEG),N-(2-hydroxypropyl)methacrylamide (HPMA), and poly(lactide-co-glycolide)(PLGA) copolymers.

The polymer of the conjugate, for example PEG or albumin, may act, orserve, to inhibit passage of the antagonist across the blood brainbarrier. As used herein the term “inhibit”, whether in this or any othercontext, includes reducing as well as preventing.

Many different classes of GnRH antagonist are known, including but notlimited to peptide or polypeptide/protein-based antagonists andnon-peptide small molecule organic compounds in a number of differentchemical classes. A “small molecule” antagonist is defined herein as anon-peptide compound of size less than 2000 Da, more particularly lessthan 1500 or 1000 Da. Examples include relugolix, elagolix,spiroindoline derivatives and ASP1707.

A “peptide GnRH antagonist” is typically an analogue of the GnRHdecapeptide, and may comprise one or more amino acid modificationsand/or substitutions. A peptide GnRH antagonist may thus comprise apeptide chain, and may comprise one or more non-native or modified aminoacids. Typically such an antagonist is 9 or 10 amino acids long, but maybe shorter or longer. A number of such antagonists are known asdescribed further below (e.g. degarelix, abarelix, ozarelix, cetrorelix,ganirelix).

The antagonists may be long acting or short-acting. A “long acting”antagonist may be defined as having a prolonged duration of action whenadministered to a subject in a single dose, e.g. at least 7, 12, 14, 15,20, 30, 40, or 60 days, or at least 2, 4, 6, or 8 months. Exemplary longacting antagonists include the peptide antagonists degarelix, abarelixand ozarelix. By corollary, a “short acting” antagonist may have aduration of action of less than 7 days when administered to a subject ina single dose, more particularly less than, 6, 5, 4, 3, 2 days or 1 day.Exemplary short-acting GnRH antagonists include peptide antagonists suchas cetrorelix and ganirelix, as well as non-peptide small moleculeantagonists such as relugolix, elagolix, spiroindoline derivatives andASP1707. A prolonged duration of action may also be achieved byformulating the antagonist as a sustained release or “depot” preparationor conjugate, e.g. with a protein such as albumin or salts and esters ofacid derivatives, according to principles and procedures known in theart. As used herein, the term “subject” refers to any animal (e.g. amammal), including, but not limited to, humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

As used herein, the term “non-human animals” refers to all non-humananimals including, but not limited to, vertebrates such as rodents,non-human primates, ovines, bovines, ruminants, lagomorphs, porcines,caprines, equines, canines, felines, ayes, etc.

As used herein, the term “sample” is used in its broadest sense. In onesense, it is meant to include a specimen or culture obtained from anysource, as well as biological and environmental samples. Biologicalsamples may be obtained from animals (including humans) and encompassfluids, solids, tissues, and gases. Biological samples include bloodproducts, such as plasma, serum and the like. Environmental samplesinclude environmental material such as surface matter, soil, water,crystals and industrial samples. Such examples are not however to beconstrued as limiting the sample types applicable to the presentinvention.

As used herein, the term “drug” is meant to include any molecule,molecular complex, prodrug, or substance administered to an organism fordiagnostic or therapeutic purposes, including medical imaging,monitoring, contraceptive, cosmetic, nutraceutical, pharmaceutical andprophylactic applications. The term “drug” is further meant to includeany such molecule, molecular complex or substance that is chemicallymodified and/or operatively attached to a biologic or biocompatiblestructure.

As used herein, the term “purified” or “to purify” or “compositionalpurity” refers to the removal of components (e.g. contaminants) from asample or the level of components (e.g. contaminants) within a sample.For example, unreacted moieties, degradation products, excess reactants,or byproducts are removed from a sample following a synthesis reactionor preparative method.

The terms “test compound” and “candidate compound” refer to any chemicalentity, pharmaceutical, drug, and the like that is a candidate for useto treat or prevent a disease, illness, sickness, or disorder of bodilyfunction (e.g. cancer). Test compounds comprise both known and potentialtherapeutic compounds. A test compound can be determined to betherapeutic by screening using screening methods known in the art.

The terms “peripheral GnRH” or “inflammatory peripheral GnRH” mean thatthe subject has a GnRH level in the periphery of the body, that isoutside the brain, which is elevated, or particularly elevated ascompared with a reference subject does not have peripheral GnRH, or ascompared to a normal reference peripheral GnRH (e.g. 0 to 160 pg/ml).Elevated peripheral levels of GnRH may be associated with inflammationor with an inflammatory condition (e.g. they may be indicative of aninflammatory condition, or may predispose to or cause or lead orcontribute to an inflammatory condition), in particular with age-relatedinflammation, as part of the expected ageing process. Accordingly,increased levels of peripheral GnRH are proposed to be inflammatory(hence the use of the term “inflammatory peripheral GnRH”). A subjectwith peripheral GnRH may have a low level systemic inflammation (that isa generalised inflammation throughout the body), e.g. a chronicinflammation, but does not necessarily exhibit signs or symptoms ofinflammatory disease. The subject may be healthy. More particularly,such a subject may have a level of peripheral GnRH which is 160 pg/ml orabove, e.g. in the plasma or serum. Whilst not wishing to be bound bytheory, the GnRH may be secreted by immune cells, specificallyperipheral immune cells, for example T-cells. The GnRH may act upon theT-cells in a cytokine-like way, stimulating T-cell proliferation andmaturation. GnRH may also act on B cells. A GnRH antagonist may act tocombat peripheral inflammation by inhibiting the action of GnRH onimmune cells, e.g. T and/or B cells, e.g. by inhibiting the effect ofGnRH on GnRH receptors on such cells.

“Chronic inflammation” means an inflammation (e.g. an inflammatorycondition) that is of persistent or prolonged duration in the body of asubject. Generally speaking this means an inflammatory response orcondition of duration of 20, 25 or 30 days or more or 1 month or more,more particular of at least 2 or 3 months. Chronic inflammation leads toa progressive shift in the type of cells present at the site ofinflammation. Chronic inflammation may occur as a result of persistentor prolonged injury or infection, prolonged exposure to toxic substancesor by autoimmune responses or conditions. Chronic inflammation may be afactor in the development of a number of diseases or disorders,including particularly degenerative diseases, or diseases or conditionsassociated with loss of youthful function or ageing (e.g. as discussedabove).

“Systemic inflammation” is inflammation which is not confined to aparticular tissue or site or location in the body. The inflammation maybe generalised throughout the body. Systemic inflammation typicallyinvolves the endothelium and other organ systems.

“Low-level inflammation” (which term is used herein as synonymous with“low-grade inflammation”) is characterised by a 2- to threefold increasein the systemic concentrations of cytokines such as TNFα, IL-6 and CRP,e.g. as measured in the plasma or serum. The increase may be relativeto, or as compared with, normal concentrations or referenceconcentrations, for example concentrations as determined in a particularreference cohort or population of subjects, e.g. young subjects (e.g.young adults) or healthy subjects, for example subjects who are notsuffering from any disease or condition, including any inflammatorydisease, or who do not have inflammation. The increase may also berelative to the level of concentration in a subject prior to developmentof the inflammation. Low-level inflammation may be observed in theabsence of overt signs or symptoms of disease. Thus, low-levelinflammation may be sub-clinical inflammation. Alternatively, a subjectwith low-level inflammation may not have a clinically diagnosedcondition or disease, but may exhibit certain signs or symptoms of aninflammatory response or inflammatory condition. In other words, theremay be signs or symptoms of the effect of inflammation in the body, butthis may not yet have progressed to an overt or recognised disease.Low-level inflammation may be peripheral inflammation, that is moreparticularly inflammation associated with peripheral GnRH, as discussedabove.

“Age-related inflammation” (or “age-associated inflammation”) is aninflammation, typically a chronic, particularly a chronic systemicinflammation which occurs with increasing age. Such inflammation may beobserved above the age of 30, 35 or 40 but typically is seen in subjectsaged 45, 50, 55 or 60 or more. In many cases this may be a low-levelinflammation.

“Cancer inflammation” is inflammation that occurs in the context ofcancer and may alternatively be defined as “cancer-associatedinflammation”. Inflammation has been identified as a hallmark of cancerand may be necessary for tumorgenesis and maintenance of the cancerstate. Cancer symptoms are associated with inflammation. Thus a subjectwith cancer may have or exhibit inflammation, which can be a low-levelor peripheral inflammation as discussed above, and in particular achronic or systemic inflammation as discussed above.

“Long-term” administration means that the GnRH antagonist isadministered for a period of at least 12 weeks. This includes that theGnRH antagonist is administered such that it is effective over, or for,a period of at least 12 weeks and does not necessarily imply that theadministration itself takes place for 12 weeks, for example if sustainedrelease compositions or long acting antagonists are used. Thus, thesubject is treated for a period of at least 12 weeks. In many cases,long-term administration is for at least 4, 5, 6, 7, 8, 9 months ormore, or for at least 1, 2, 3, 5, 7 or 10 years, or more.

A “biologic” drug or agent, for example as typically used to treat aninflammatory disease such as rheumatoid arthritis, is any agent which isderived from, based on, or comprises a biological molecule. Typically,this may be an antibody, which term includes monoclonal and polyclonalantibodies, antibody fragments, and antibody derivatives such as e.g.chimeric humanised antibodies or single chain antibodies etc., or aanother protein such as a receptor or receptor chain, domain orfragment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to screening, diagnosis, prognosticevaluation, and treatment or prevention of an inflammatory conditionselected from age-related inflammation, chronic inflammation, andinflammatory diseases. In particular, the present invention relates tomethods of treating inflammatory diseases (e.g., rheumatoid arthritis orspondyloarthritis) or patients with inflammatory peripheral GnRH withdrugs that lower the effects of GnRH or GnRH inhibitors.

The stimulation of the hypothalamic-pituitary-gonadal (HPG) axis isrelated to systemic aging and lifespan. It has been shown thatage-related hypothalamic changes occur independently of changes ingonadal hormones. GnRH pulse amplitude is increased and particularlyerratic during the menopausal transition, when the risk forcardiovascular disease and osteoporosis is accelerated in females.Therefore, one may speculate that the pronounced rapid changes in GnRHand gonadotropin levels might be of particular importance in not onlythe pathogenesis of autoimmune diseases, where early menopause has beenshown to be a risk factor, but also in the pathogenesis ofcardiovascular disease and osteoporosis. This is also the time of thegreatest and most erratic GnRH pulse amplitude as well as the greatestrate of unfavourable changes in lipid markers, and the greatest rate ofbone mineral density loss in females.

GnRH is transported in a unique hypothalamic portal system and israpidly degraded after reaching the pituitary. It has been suggestedthat the isolated hypophyseal portal system may not only have evolvedsolely as a means to deliver hypothalamic peptides to the pituitary, butalso as a way to prevent their delivery to extra-pituitary targets. Thisis in accordance with a detrimental inflammatory GnRH.

There is a need for better treatments of inflammatory diseases, chronicinflammation, and therefore age-related inflammation.

The manuscript by Kass et al., 2014 Scand. J. Rheumatol., 43 (1), 22-27,describes a short term study of a GnRH antagonist, Cetrorelix (AGRAstudy). This is the first human study of GnRH antagonist treatment in aninflammatory disease. The study was conducted over a period of 5 daysonly and did not meet the primary end-point with regard to clinicalresponse. However, some patients did show some response and the responsewas thought to be limited to patients with above normal levels of LH andFSH (Abstract to the 13^(th) annual American College of Rheumatologymeeting). The study did show a possible reduction of TNFα. However,current TNFα inhibiting therapy in rheumatoid arthritis is long term.The fact that TNFα is acutely reduced by GnRH antagonists, as indicatedin the AGRA study, does not show that GnRH antagonism is beneficial inrheumatoid arthritis or other inflammatory diseases using long termtherapy. There are several examples in medicine that give positiveimmediate effects in disease but are unsuitable for long term use,either due to lack of efficacy, safety or both.

Prior to the present disclosure, a person skilled in the art would lookfor further data on the long term safety of GnRH antagonism inpremenopausal females, postmenopausal females, and males. Degarelix, adepot GnRH antagonist, has only been tested in males over long term.Furthermore, cetrorelix is actually contraindicated in postmenopausalfemales. In contrast, the present disclosure, in some embodiments,encompasses the long term use of GnRH antagonists in males,postmenopausal females, as well as premenopausal females. Examples ofbeneficial uses of medicines in rheumatoid arthritis over short terminclude high dose non-steroidal anti-inflammatory drugs or high doseintravenous steroids. These are treatments that must not be used overlong term due to serious side effects. Side effects of GnRH antagonisttherapy are believed to be osteoporosis and cardiovascular disease. Aspatients with rheumatoid arthritis are much more prone to osteoporosisand cardiovascular disease due to their systemic inflammation, a personskilled in the art would not attempt to treat these patients with longterm GnRH antagonist therapy without further safety data, which thepresent disclosure provides. Furthermore, it is widely accepted that thereduction of oestrogen reduces bone density and contributes tocardiovascular disease. Therefore GnRH antagonists, which inhibitoestrogen, are expected to reduce bone density and contribute tocardiovascular disease.

Prior to the present disclosure, a person skilled in the art would havealso looked for further data on the long term efficacy of GnRHantagonism in premenopausal females, postmenopausal females, and males.Maintenance doses for GnRH lowering drugs would be unknown, withoutfurther experimentation. The AGRA study was based upon a previous study(Kass et al., 2010, Scand. J. Rheumatol. 39 (2), 109-117), whichemphasized that the relative reduction of LH is important in improvingdisease activity. The study showed that low LH levels were notassociated with low disease activity. That study also showed there wasno association with absolute LH levels and disease activity, onlyrelative changes of hormones. Therefore, one does not expect that stablylow LH levels can show continued improvements in rheumatoid arthritis.There is no prior evidence to show that stably low levels of LH can bebeneficial in rheumatoid arthritis. A relative reduction in LH can onlybe demonstrated at the initiation of therapy e.g. first 1-5 days.Indeed, that is why the AGRA trial was designed to give 5 days oftherapy, for the greatest relative change in LH. Thereafter, withcontinued GnRH antagonist administration, levels will be stably low.

The AGRA study showed some short term anti-inflammatory effects inrheumatoid arthritis. However, the long term data with degarelix,cetrorelix and ganirelix treatment in patients shows surprising unlikelyimprovements in severely ill patients. The mean disease activity scoreof the AGRA patients was 5.0, whereas the mean degarelix diseaseactivity score was 7.3 (scale 1.3-8.8, with higher numbers denotinggreater disease activity); this supports a substantially greater diseaseactivity in the degarelix patients compared to patients in the AGRAstudy. Due to these baseline differences, the effects of degarelix aresurprising compared to the short term study.

Cetrorelix is used to treat hormone-sensitive cancers of the prostateand breast, and some benign gynaecological disorders. In addition, it isused in assisted reproduction to inhibit premature LH surges. The drugblocks the action of GnRH upon the pituitary, thus rapidly suppressingthe production and action of LH and FSH. Both cetrorelix and ganirelixare administered as 0.25 mg daily s.c. injections. Degarelix is a depotmonthly GnRH antagonist injection licensed for prostate cancer in maleswith a loading dose of 240 mg, and 80 mg monthly injections thereafter.The present disclosure provides different dosing schedules for patientswith chronic inflammation.

Through studying long term GnRH antagonist treatment, we have alsoidentified not only that stably low LH levels show continuedimprovements, but also any increase in LH, for example due to longintervals between degarelix injections, can cause flares in rheumatoidarthritis (RA) patients. Therefore, in some embodiments, LH levels staylow throughout long term therapy.

The present disclosure provides the unexpected result that rheumatoidarthritis patients and other subjects with an inflammatory condition canbe treated safely with a GnRH antagonist over the long term. Experimentsdescribed herein provide an example of a patient (patient 6 inExample 1) who did not improve in the short term study of 5 days, butimproved surprisingly with long term treatment. The definition of longterm treatment is treatment over 12 weeks according to FDA guidelines(Guidance for Industry Rheumatoid Arthritis: Developing Drug Productsfor Treatment, May 2013). The disclosure also shows that long termtherapy is safe and effective in postmenopausal females (previouslycontraindicated), premenopausal females, and males, without contributingto increased cardiovascular disease or osteoporosis as demonstrated bybone scans, laboratory variables and blood pressure, sometimes incombination with individual titration to baseline or higher levels ofoestradiol or testosterone.

The disclosure further shows that stably low levels of LH contribute tocontinued improvements in rheumatoid arthritis.

Rheumatoid arthritis may develop, flare, or subside during hormonalchanges in the HPG axis; for example, during pregnancy, postpartum,menopause, or aromatase inhibition therapy. These observations haveprompted research into the effects of gonadal hormones of the HPG axis,such as oestrogen and testosterone in rheumatoid arthritis; but theresults have been inconclusive.

Hypothalamic and pituitary hormones of the HPG axis control gonadalhormones. Gonadal hormones in both sexes are stimulated by pituitary LHand FSH. LH and FSH secretion are stimulated by the hypothalamic GnRH.GnRH, LH, and FSH have important physiological roles in both male andfemale reproduction. Therefore, these hormones may be involved inpathological processes in males as well as females.

Experiments conducted during the course of development of embodiments ofthe present invention demonstrated that GnRH-antagonism producedsustained long term anti-inflammatory effects in rheumatoid arthritispatients. Further experiments demonstrated that GnRH antagonists can beused to lower the amount of cytokines such as TNFα, IL-1β, IL-10, andIL-2. Furthermore, the inventor shows that GnRH antagonists reduce thelevels of the acute phase protein CRP. Accordingly, embodiments of thepresent invention provide methods and uses of treating an autoimmune orinflammatory disease, comprising administering a GnRH antagonist to thesubjects.

In one embodiment, the subjects are patients suffering rheumatoidarthritis. In some embodiments, subjects to be treated do not respond tomethotrexate. In some embodiments, subjects to be treated do not respondto anti-TNF treatment. In some embodiments, the subjects to be treateddo not respond to biologics as described above.

In some embodiments, subjects are women (e.g. postmenopausal women orwomen over age 40). In some embodiments, women are treated with a GnRHantagonist at a specific point in the menstrual cycle (e.g. mid-cyclewhen LH and FSH levels reach a high point). While not limited to aparticular mechanism, it is contemplated that such treatment preventspremenstrual flare ups of RA symptoms. In some embodiments, subjects aremen over age 40 (e.g. over age 50, 60, or 70).

In some embodiments, the patient population is defined as negative forCCP antibodies. In some embodiments, the patient population is definedas DMARD and/or TNF non responders.

Further experiments demonstrated that GnRH antagonists find use in thetreatment and prevention of systemic lupus erythematosus, nephritisincluding lupus nephritis, ankylosing spondylitis, multiple sclerosis,scleroderma, psoriasis, inflammatory bowel disease, osteoporosis (e.g.by increasing bone mineral density), and cardiovascular disease (e.g. bydecreasing HbA1c, decreasing blood pressure, or increasing HDL levels).

In some embodiments, the risk of cardiovascular disease is estimated ina variety of ways by a number of prognostic indicators. The FraminghamRisk Score is based on data obtained from the Framingham Heart Study andis used to estimate the 10-year cardiovascular risk of an individual.The Framingham Risk Score is a calculated estimated risk for developingfatal or non-fatal cardiovascular event based on a composite score basedon a pre-existing risk factors, including: age, gender, systolic bloodpressure level (+/−treatment), HDL cholesterol level, and smoker status.A patient's risk score gives an indication of the likely benefits ofprevention and also can be a useful metric to determine the effects oftreatments.

The present invention is not limited to a particular GnRH antagonist oragent that alters the biological activity of GnRH. Examples include, butare not limited to, cetrorelix, elagolix, ganirelix, abarelix,degarelix, detirelix, iturelix, ozarelix, prazarelix, ramorelix,teverelix, elagolix, relogolix, or ASP1707. In some embodiments thepresent invention applies to any drug that prevents or inhibits GnRHreceptor signaling. GnRH inhibitors, GnRH vaccines such as GnRH-DT (theGnRH decapeptide linked to diphtheria toxoid), selective immune cellGnRH receptor antagonists, anti-GnRH antibodies, monoclonal antibodiesagainst GnRH, circulating GnRH receptor fusion proteins, spiroindolinederivatives as gonadotropin-releasing hormone receptor antagonists,non-peptide oral GnRH antagonists, kisspeptin antagonists, oestrogencompounds, testosterone compounds, LH compounds or FSH compounds,hypothalamic hormones or neuropeptides. In some embodiments, a suitableGnRH antagonist also prevents or inhibits GnRH receptor signaling. Theantagonists may be used singly or in any combination.

As mentioned above, a wide variety of different GnRH antagonists areknown and have been described in the literature, including both peptideand non-peptide antagonists, the latter including antagonists in a largeand varied range of different chemical classes. With regard tonon-peptide small molecule GnRH antagonists reference may be made to thereviews by Heitman and Ijzerman, 2008, Med. Res. Rev., 28 (6), 975-1011and Zhu and Chen, 2004, Expert Opin. Ther. Patents, 14 (2), 187-199,which reviews and the reference documents cited therein are allincorporated herein by reference.

Any of the GnRH antagonists known and described in the literature may beused. Known peptide antagonists includeacetyl-β-[2-naphthyl]-D-Ala-D-p-chloro-Phe-β-[3-pyridyl]-D-Ala-Ser-Nε-[Nicotinoyl]-Lys-Nε-{Nicotinoyl]-D-Lys-Leu-Nε-[isopropyl]-Lys-Pro-D-Ala-NH₂(Antide), acetyl D2Nal1, D4C1Phe2, D3Pal3, Arg5, Dglu6 (AA) (also knownas NaIGlu),acetyl-D2NaI,D4CIPhe-D3Pal-Ser-Aph(Ac)-D-Aph(Ac)-Leu-Lys(lpr)-Pro-D-Ala-NH₂(Abarelix, Praecis, Mass. US), Ganirelix (Orgalutron/Antagon) (Organon,West Orange, N.J.), Cetrorelix (ASTA Medica AG, Frankfurt, Germany),Cetrotide, Azaline B, Acyline (Ac-D2Nal-D4Cpa-D3Pal-Ser4-Aph(Ac)-D4Aph(Ac)-Leu-ILys-Pro-DAla-NH2), long-acting GmRHanalogues incorporating p-ureido-phenylalanines at positions 5 and 6(such as Degarelix (Ferring, Geneva, Switzerland)), FE200486,Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(L-hydroorotyl)-D4Aph(carbamoyl)-Lei-ILys-Pro-DAla-NH2 (theacetate salt of which is FE200486),Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(Atz)-D4Aph(Atz)-Leu-ILys-Pro-DAla-NH2wherein Atz is 3′-amino-1H-1′,2′,4′-triazol-5′-yl,5, the antagonistsdescribed in U.S. Pat. Nos. 5,434,136, 6,156,772, 6,156,767, 6,150,522,6,150,522, 6,150,352, 6,147,088, 6,077,858, 6,077,847, 6,025,366,6,017,944, 6,004,984, 5,998,432, and the GnRH antagonist described inPoster Sessions, Endo '98, p. 265. GnRH antagonists useful in thepresent invention may have a binding affinity that parallels theantagonistic properties and can be linear or cyclized pentapeptides todecapeptides. Of the linear peptide antagonists, peptides with largesubstitutions in position 6, such as those found in degarelix, or withlarge substitutions such as iodinated substitutions, lead to highbinding affinity.

GnRH antagonists are also described in e.g. U.S. Pat. No. 5,470,947, WO89/01944; U.S. Pat. No. 5,413,990; U.S. Pat. No. 5,300,492; U.S. Pat.No. 5,371,070, U.S. Pat. No. 5,296,468; U.S. Pat. No. 5,171,835; U.S.Pat. No. 5,003,011; U.S. Pat. No. 4,431,635; U.S. Pat. No. 4,992,421;U.S. Pat. No. 4,851,385; U.S. Pat. No. 48,015; and U.S. Pat. No.4,689,396.

In another embodiment, the gonadotropin-releasing hormone antagonist isa peptide, characterized by the structure:Ac-D-Nal-4-Cl-Phe-D-Pal-Ser-Tyr-D-Pal(N—O-Leu-Lys(iPr)-Pro-D-Ala-NH₂, orin another embodiment comprising a structure ofAc-D-Nal-4-Cl-D-Phe-D-Pal-Ser-Tyr-D-Pal(CH2-COO—)-Leu-Lys(iPr)-Pro-Ala-NH₂,or in another embodiment,Ac-Sar-4-Cl-D-Phe-D-Nal-Ser-Tyr-D-Pal(Bzl)-Leu-Lys(iPr)-Pro-Ala-NH₂ or apharmaceutically acceptable sale thereof.

U.S. Pat. No. 5,516,887 describes antarelix ([Ac-D-2Nal¹, D-4CIPhe², D3Pal³, D-N^(ε)-carbamoyl Lys⁶, Ilys⁸, D-Ala¹⁰]-GnRH. U.S. Pat. No.5,296,468 discloses the design and synthesis of a number of GnRHantagonists wherein the side chains of selected residues are reacted tocreate cyanoguanidino moieties, some of which subsequently spontaneouslyconvert to a desired heterocycle, e.g. a 3-amino-1,2,4-triazole(atz).Such cyanoguanidino moieties are built upon the omega-amino group in anamino acid side chain, such as lysine, ornithine, 4-amino phenylalanine(4Aph) or an extended chain version thereof, such as 4-aminohomophenylalanine (4Ahp). GnRH antagonists having such significantlymodified or unnatural amino acids in the 5- and 6-positions exhibit goodbiological potency, and those build upon Aph are generally considered tobe particularly potent.

Another example is Azaline B, i.e. [Ac-D-2Nal¹, D-4ClPhe², D-3 Pal³,4Aph(atz)⁵, D-4Aph(atz)⁶, ILys⁸, D-Ala¹⁰]-GnRH. U.S. Pat. No. 5,506,207discloses GnRH antagonists with acylated, amino-substitutedphenylalanine side chains of residues in the 5- and 6-positions; onesuch decapeptide is Acyline, [Ac-D-2Nal¹, D-4CIPen², D-3 Pal³,4Aph(Ac)⁵, D-4Aph(Ac)⁶, ILys⁸, D-Ala¹⁰]-GnRH.

Peptide antagonists generally may be subject to degradation in the GItract and so tend to be administered parenterally, typically byinjection, e.g. subcutaneously or intramuscularly.

As regards small molecule-peptide GnRH antagonists, at least 14different chemical classes of compounds have been reported. Many suchantagonists have the advantage that they may be administered orally. Thechemical classes include thieno[2,3-d]pyridin-4-one derivatives,quinolin-2-one derivatives, indole derivatives, pyrrolo[1,2-a]pyrimid-7-one derivatives, imidazolo[1,1-a]pyramidin-5-onederivatives, thieno[2,3-d]pyrimidin-2,4-dione derivatives, furamidederivatives, pyrimidin-2,4-dione derivatives, benzimidazole derivatives,1,3,5-triazine-2,4,6-trione derivatives,thiazolino[3,2-c]pyramidin-5,7-diones and oxazole derivatives thereof,tetrahydropyrido[4,3,d]pyrimidin-2,4-dione derivatives,tetrahydropyrrolo[3,2-c]pyridines, thieno[2,3-b]pyrolle derivatives,3-pyrazinone, pyrid-2-one and pyrid-4-one derivatives, various otherpyrazole and pyrrole derivatives, oxazole- and thiazole-4-carbamidecompounds, tetrahydroisoquinoline derivatives. 1,3-dihydrobenzimidazolederivatives, imidazo[1,2-a]pyridines, bicyclic pyrrolidines, quinolines,imidazo[4,5-c]pyridines, benzimidazoles, benzoxazoles, benzothiazoles,quinazoline-2,4-diones, tricyclic pyrrolidines, 1,2,3,4-tetrahydrocarbazoles. More recently, certain spiroindoline derivatives have beenfound to have GnRH antagonist activity. GnRH receptor antagonists havethus been described with a wide range of diverse chemical structures.Spiroindoline GnRH antagonists are described in WO2014166958 (Bayer),which is incorporated herein by reference. Representative examples ofsuch spiroindoline compounds include compounds of the following Formula1:

The compounds may be prepared as racemic mixtures and resolved into twoenantiomers by using chiral column chromatography methods.

Reference may also be made to the following exemplary patentapplications describing the various different GnRH antagonists, whichalso incorporated herein by reference: WO 95/28405, WO 97/14697, WO97/41126, WO/000 0493, WO96/24597, WO00/56739, WO99/33831, U.S. Pat. No.6,413,972 (Takeda); WO99/21557, WO99/41252, WO0069433, WO99/51231,WO99/51595, WO00/53602, WO97/44037, WO01/0228 (Merck and Co);WO00/69859, WO015519, WO03/011293, WO 03/011841, WO03011870 (NeurocrineBioscience Inc); WO99/44987 (Alanex Corp.); WO03/06879 (Agouron Pharm.Inc); WO02/092565, WO 02/066478, WO02/066477 (AstraZeneca); WO02/11732(Glaxo Group Ltd); WO02/066437 (Schering); WO02/48112 (OrthoMcNeilPharm. Inc); WO03/053939 (SCRAS); WO02/02533 (Yamanouchi Pharm. Co).

Many GnRH antagonists may be obtained commercially. Degarelix ismarketed under the name Firmagon by Ferring. Ganirelix is described inU.S. Pat. No. 5,767,082, and U.S. Pat. No. 6,653,286 and is availablefrom Merck/MSD. Cetrorelix is available from Merck Serono. Relugolix isavailable from Takeda. Elagolix is available from Abbvie/NeurocrineBiosciences Inc. Spiroindoline derivatives are available from Bayer. AGnRH-DT vaccine is available from GSK. Various pyrazole and pyrrolecompounds are available from AstraZeneca.

ASP1707 is a benzimidazoylidene propane-1,3-dione derivative. It andother propane-1,3-dione derivatives are described in WO 2005/118556,U.S. Pat. No. 8,076,367,U.S. Pat. No. 7,569,688 and U.S. Pat. No.7,960,562, the entire contents of which are incorporated herein byreference. In particular, ASP1707 is described in Example 726 of U.S.Pat. No. 7,960,562, and has the formula(2R)—N-({5-[3-(2,5-difluorophenyl)-2-(1,3-dihydro-2H-benzoimidazol-2-ylidene)-3-oxopropanoyl]-2-fluorophenyl}sulfonyl)-2-hydroxypropanimidamide.Its structure is shown below. ASP1707 is available from Astellas, andcan also be manufactured according to the methodology described inExample 696 of U.S. Pat. No. 7,960,562. In this instance, ASP1707 issynthesised by subjecting (2R)-2-hydroxypropanimidamide hydrochloride toa sulfonyl amidination reaction with5-[3-(2,5-difluorophenyl)-2-(1,3-dihydro-2H-benzoimidazol-2-ylidene)-3-oxopropanoyl]-2-fluorophenylsulfonylchloride in the presence of sodium hydride in tetrahydrofuran, followedby a liquid separation and purification with columns. Other methods ofproducing ASP1707 are disclosed in US 2015/0259299, the entire contentof which is also incorporated herein by reference.

In any of the methods and uses according to the invention hereindescribed, any pharmaceutically acceptable salt of ASP1707 may be usedinterchangeably with ASP1707 itself. Such salts may includeacid-addition salts with an inorganic acid, such as hydrochloric acid,sulphuric acid, nitric acid, phosphoric acid, etc., and acid additionsalts with an organic acid such as formic acid, acetic acid, propionicacid etc. Such salts may also include salts with an inorganic base, suchas sodium, potassium or calcium etc., or an organic base such as lysine,ornithine, ethylamine etc.

ASP1707:

In some embodiments, a suitable ‘GnRH antagonist’ may be too large tocross the blood-brain barrier (BBB). As described above, in oneembodiment such large antagonists may be provided by coupling orconjugating a GnRH receptor antagonist to a polymer, includingparticularly a polymer selected from a polypeptide, a polysaccharide, apolyethylene glycol or HPMA or a PLGA copolymer. The polypeptide maytypically be a protein such as albumin, or the Fc part of an antibody.The polysaccharide may for example be a dextran etc. Thus such a non-BBBcrossing GnRH antagonist may include PEGylated GnRH inhibitors, or GnRHinhibitors fused to proteins such as albumin. These conjugates may beadministered via several routes, including but not limited tosubcutaneous and oral routes.

Accordingly, any of the GnRH antagonists described above may be coupledto a polymer, including particularly peptide antagonists such asdegarelix, cetrorelix or ganirelix, or small molecule non-peptideantagonists such as elagolix, relugolix, ASP1707, or a spiroindolinederivative.

Such conjugates represent a novel aspect of the present invention.Accordingly, in a further aspect the present invention provides aconjugate comprising a GnRH antagonist linked to a polymer, moreparticularly a polymer which serves (more particularly specificallyserves) to inhibit passage of the GnRH antagonists across the BBB. Sucha conjugate may be used for the treatment or prevention of anyinflammatory condition, as defined and discussed above.

In a further aspect, the invention accordingly provides such a conjugatefor use in therapy.

In a still further aspect, the invention provides a pharmaceuticalcomposition containing a conjugate of the invention as herein defined,together with at least one pharmaceutically acceptable carrier orexcipient.

Such GnRH conjugates may readily be prepared using well known proceduresand reagents, as described in the art. Thus, a variety of differentmethods and reagents for linking peptides or non-peptide small organicmolecules to polymers such as proteins or other polypeptides,polysaccharides or polyethylene glycol are available and would be knownto the person skilled in this art. The GnRH antagonists may be linkeddirectly or indirectly, e.g. via a spacer or linker group, to thepolymer.

The conjugates of the invention have particular utility in long termtreatment of an inflammatory condition, but are not limited to such use.In a particular embodiment they have utility in the treatment of chronicinflammation, age-related inflammation or inflammatory peripheral GnRH.In this regard, such conjugates are advantageous in that by not beingable to cross the BBB, they do not act centrally (in other words theyare inhibited from acting at central GnRH receptors, that is GnRHreceptors on the pituitary). In this way undesirable side-effects may beavoided, and in particular side-effects comprising or involvinginhibition of sex hormone (e.g. oestrogen or testosterone) productionand/or action.

It would be highly advantageous if one could only access peripheral GnRHreceptors without disturbing central GnRH receptors. For long termtreatment, e.g. of several years, in e.g. chronic inflammation, thisavoids side effects of decreasing FSH, LH, oestrogen, and testosterone.Therefore, one avoids disrupting menstrual cyclicity in premenopausalwomen, in order to maintain fertility. Prior to the present disclosureone would have anticipated that GnRH antagonists should only workcentrally on pituitary gonadotropes. The present disclosure describesthe treatment of inflammatory peripheral GnRH, that contributes tochronic inflammation and age associated inflammation. This feature ofGnRH inhibition is possible through drugs that lower the peripheraleffects of GnRH without crossing the BBB. Such compounds would have tobe larger than the currently available GnRH antagonists which do crossthe BBB. Examples include, but are not limited to, a larger GnRHantagonist such as a GnRH antagonist attached to albumin, or a PEGylatedGnRH antagonist, which are producible by persons skilled in the art. Thesame may be applied to other hormones, peptides, or substances that areinvolved in the regulation of GnRH including, but not limited to,kisspeptin. Various modifications and variations of the described methodand system of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention.

The present invention further provides the use of a GnRH antagonist inthe treatment of an autoimmune disease in a subject.

The present invention is not limited to the treatment of a particularautoimmune or inflammatory disease. In some embodiments, the disease isrheumatoid arthritis. In some embodiments, inflammatory diseases includebut are not limited to arthritis, inflammatory bowel disease, psoriaticarthritis, osteoarthritis, degenerative arthritis, polymyalgiarheumatic, ankylosing spondylitis, reactive arthritis, gout, pseudogout,inflammatory joint disease, systemic lupus erythematosus, polymyositis,and fibromyalgia. Additional types of arthritis include achillestendinitis, achondroplasia, acromegalic arthropathy, adhesivecapsulitis, adult onset Still's disease, anserine bursitis, avascularnecrosis, Behcet's syndrome, bicipital tendinitis, Blount's disease,brucellar spondylitis, bursitis, calcaneal bursitis, calciumpyrophosphate dihydrate deposition disease (CPPD), crystal depositiondisease, Caplan's syndrome, carpal tunnel syndrome, chondrocalcinosis,chondromalacia patellae, chronic synovitis, chronic recurrent multifocalosteomyelitis, Churg-Strauss syndrome, Cogan's syndrome,corticosteroid-induced osteoporosis, costosternal syndrome, CRESTsyndrome, cryoglobulinemia, degenerative joint disease, dermatomyositis,diabetic finger sclerosis, diffuse idiopathic skeletal hyperostosis(DISH), discitis, discoid lupus erythematosus, drug-induced lupus,Duchenne's muscular dystrophy, Dupuytren's contracture, Ehlers-Danlossyndrome, enteropathic arthritis, epicondylitis, erosive inflammatoryosteoarthritis, exercise-induced compartment syndrome, Fabry's disease,familial Mediterranean fever, Farber's lipogranulomatosis, Felty'ssyndrome, Fifth's disease, flat feet, foreign body synovitis, Freiberg'sdisease, fungal arthritis, Gaucher's disease, giant cell arteritis,gonococcal arthritis, Goodpasture's syndrome, granulomatous arteritis,hemarthrosis, hemochromatosis, Henoch-Schonlein purpura, Hepatitis Bsurface antigen disease, hip dysplasia, Hurler syndrome, hypermobilitysyndrome, hypersensitivity vasculitis, hypertrophic osteoarthropathy,immune complex disease, impingement syndrome, Jaccoud's arthropathy,juvenile ankylosing spondylitis, juvenile dermatomyositis, juvenilerheumatoid arthritis, Kawasaki disease, Kienbock's disease,Legg-Calve-Perthes disease, Lesch-Nyhan syndrome, linear scleroderma,lipoid dermatoarthritis, Lofgren's syndrome, Lyme disease, malignantsynovioma, Marfan's syndrome, medial plica syndrome, metastaticcarcinomatous arthritis, mixed connective tissue disease (MCTD), mixedcryoglobulinemia, mucopolysaccharidosis, multicentricreticulohistiocytosis, multiple epiphyseal dysplasia, mycoplasmalarthritis, myofascial pain syndrome, neonatal lupus, neuropathicarthropathy, nodular panniculitis, ochronosis, olecranon bursitis,Osgood-Schlatter's disease, osteoarthritis, osteochondromatosis,osteogenesis imperfecta, osteomalacia, osteomyelitis, osteonecrosis,osteoporosis, overlap syndrome, pachydermoperiostosis Paget's disease ofbone, palindromic rheumatism, patellofemoral pain syndrome,Pellegrini-Stieda syndrome, pigmented villonodular synovitis, piriformissyndrome, plantar fasciitis, polyarteritis nodos, polymyalgiarheumatica, polymyositis, popliteal cysts, posterior tibial tendinitis,Pott's disease, prepatellar bursitis, prosthetic joint infection,pseudoxanthoma elasticum, psoriatic arthritis, Raynaud's phenomenon,reactive arthritis/Reiter's syndrome, reflex sympathetic dystrophysyndrome, relapsing polychondritis, retrocalcaneal bursitis, rheumaticfever, rheumatoid vasculitis, rotator cuff tendinitis, sacroiliitis,salmonella osteomyelitis, sarcoidosis, saturnine gout, Scheuermann'sosteochondritis, scleroderma, septic arthritis, seronegative arthritis,shigella arthritis, shoulder-hand syndrome, sickle cell arthropathy,Sjogren's syndrome, slipped capital femoral epiphysis, spinal stenosis,spondylolysis, staphylococcus arthritis, Stickler syndrome, subacutecutaneous lupus, Sweet's syndrome, Sydenham's chorea, syphiliticarthritis, systemic lupus erythematosus (SLE), Takayasu's arteritis,tarsal tunnel syndrome, tennis elbow, Tietse's syndrome, transientosteoporosis, traumatic arthritis, trochanteric bursitis, tuberculosisarthritis, arthritis of ulcerative colitis, undifferentiated connectivetissue syndrome (UCTS), urticarial vasculitis, viral arthritis,Wegener's granulomatosis, Whipple's disease, Wilson's disease, andyersinial arthritis.

In some embodiments, the GnRH or gonadotropin antagonist is administeredin combination with an additional treatment i.e. additional active agent(e.g. treatments or agents known to be useful or effective in thetreatment of autoimmune or inflammatory disease such as rheumatoidarthritis). Such an additional active agent may accordingly be adisease-modifying drug and in particular a disease-modifying drug foruse in treating or preventing inflammation or an inflammatory conditionas defined herein. Examples of such agents include, but are not limitedto, disease-modifying anti-rheumatic drugs (e.g. leflunomide,methotrexate, sulfasalazine, hydroxychloroquine), disease-modifyingagents for use in treating multiple sclerosis (e.g. fampridine),biologic agents (e.g. rituximab, infliximab, etanercept, adalimumab,golimumab, tofacitinib, anakinra, abatacept), nonsteroidalanti-inflammatory drugs (e.g. ibuprofen, celecoxib, ketoprofen,naproxen, piroxicam, diclofenac), analgesics (e.g. acetaminophen,tramadol), steroids and glucocorticoids (e.g. prednisone,methylprednisone), and therapies for osteoporosis such as Fosamax orzoledronic acid.

In a further aspect the invention also provides a GnRH antagonist foruse in the treatment or prevention of an inflammatory condition in asubject, selected from an inflammatory disease, chronic inflammation,age-related information or inflammatory peripheral GnRH, wherein saidGnRH antagonist is for co-administration to said subject together with afurther active agent, and in particular wherein said further activeagent is a disease-modifying drug, or a sex hormone or an agent whichregulates sex hormone production and/or activity (e.g. an agent usefulin sex hormone substitution therapy).

A sex hormone may be oestrogen or testosterone or an oestrogen ortestosterone derivative. Such oestrogen or testosterone derivatives arewidely described in the art and available commercially. Likewise agentsuseful in sex hormone substitution therapy are also well known in theart and widely available, and include for example LH or FSH or LH or FSHderivatives or analogues.

In an embodiment the further active agent may be useful in, or effectivefor, the treatment of an inflammatory condition, e.g. an inflammatorydisease including an inflammatory disease as defined herein. The furtheractive agent may be as described above. In a particular embodiment ofthis aspect of the invention, the GnRH antagonist may be for long-termadministration.

This aspect of the invention also provides kits or combined/combinationproducts containing or comprising a GnRH antagonist and an additionalactive agent. In particular, such kits or combined/combination productsare for use in treating or preventing an inflammatory condition asdefined herein.

The GnRH antagonist and additional active agent may be formulated foradministration together, e.g. in a single pharmaceutical composition, orthey may be formulated for separate, e.g. sequential or simultaneous, orsubstantially simultaneous, administration. Thus, the kit orcombined/combination product may comprise separate containers, eachcontaining a GnRH antagonist and a further active agent.

The GnRH antagonist and the additional active agent may be administeredby the same route or by different routes. Thus for example in oneembodiment the GnRH antagonist may be administered parenterally, e.g. byinjection (e.g. subcutaneous or intramuscular injection) and theadditional active agent may be administered orally. In other embodimentsboth the components may be administered orally, or both may beadministered parenterally e.g. by injection.

In a particular aspect, the invention provides a product (e.g. a kit)comprising a GnRH antagonist and an additional active agent as acombined preparation for simultaneous, separate or sequential use in thetreatment or prevention of an inflammatory condition in a subject,selected from an inflammatory disease, chronic inflammation, age-relatedinflammation or inflammatory peripheral GnRH, wherein said additionalagent is useful in the treatment of said inflammatory condition.

In a preferred embodiment said GnRH antagonist and additional activeagent are for long-term administration to said subject for a period ofat least 12 weeks.

Certain Examples included below demonstrate that the GnRH antagonistswhen administered with an additional active agent may exhibit synergy.There may be an additional, e.g. greater than cumulative, effect whenthe additional active agent is co-administered with a GnRH antagonist.In other embodiments, the clinical benefit experienced by the subjectmay be improved or augmented in any way, when the GnRH antagonist isco-administered with the additional active agent. Synergisticcombinations of a GnRH antagonist and an additional active agentrepresent one preferred embodiment of the invention.

When the condition being treated is arthritis, the additional agent canbe an agent effective in treating arthritis (e.g. TNFα inhibitors suchas anti-TNFα monoclonal antibodies (such as REMICADE® (infliximab),CDP-870 and HUMIRA™ (adalimumab) and TNF receptor-immunoglobulin fusionmolecules (such as ENBREL® (entanercept)), IL-1 inhibitors, receptorantagonists or soluble IL-1Rα (e.g. KINERET™ (anakinra) or IL-1βconverting enzyme (ICE) inhibitors), nonsteroidal anti-inflammatoryagents (NSAIDS), piroxicam, diclofenac, naproxen, flurbiprofen,fenoprofen, ketoprofen ibuprofen, fenamates, mefenamic acid,indomethacin, sulindac, apazone, pyrazolones, phenylbutazone, aspirin,COX-2 inhibitors (such as CELEBREX® (celecoxib), VIOXX® (rofecoxib),BEXTRA® (valdecoxib) and etoricoxib (preferably MMP-13 selectiveinhibitors)), NEURONTIN® (gabapentin), pregabalin, sulfasalazine, lowdose methotrexate, leflunomide, hydroxychloroquine, d-penicillamine,auranofin, parenteral or oral gold), rituximab, roactemera, or orencia).The additional agents to be co-administered can be any of the well-knownagents in the art, including, but not limited to, those that arecurrently in clinical use.

In some embodiments the GnRH antagonist is administered in combinationwith hormone substitution therapy (e.g. testosterone, such as Testogel50 mg every 1-5 days, oestrogen, such as Activelle 1 mg/0.5 mg every 1-5days, LH, such as 75IE lutropin alfa daily, or every 2-5 days, or FSH,such as 75-150IE follitropin alfa daily, or every 2-5 days.

In some embodiments, the patients receive oestrogen or testosteronesupplement daily or every 2-6 days or weekly, in addition to GnRHantagonist.

In some embodiments, the physician titrates the dose of oestrogen ortestosterone to the patient's baseline or higher levels.

Accordingly, in a representative embodiment, where the additional activeagent is a sex hormone or an agent effective for hormone substitutiontherapy, the dose of the sex hormone or other agent is titrated toachieve a desired or selected oestrogen or testosterone level in thesubject, for example a level which is substantially equal to or higherthan a baseline level, or in some cases lower than a baseline level.Such a baseline level may be the level of the subject prior toadministration of the GnRH antagonist, or it may be a reference baselinelevel, for example a level of oestrogen or testosterone which is anormal level or which is a typical level for that subject (e.g. an age-and/or sex-matched subject or a healthy subject of the appropriategender). An appropriate or desired level may be selected according toneed or circumstance, for example based on the age, sex and/or clinicalcondition of the patient. Thus, for example, as a patient ages a lowerlevel may be appropriate, particularly over a long course of treatmentover many years. Thus, the desired or appropriate level may change.

In some embodiments, the GnRH antagonist is continually administered, orgiven as a vaccination with booster doses, or administered in one ormore repeated doses (e.g. up to 6 times daily, e.g. 2, 3 or 4 to 6 timesdaily, daily, weekly, monthly, or other interval) for a period of time(e.g. at least three months, at least 6 months, at least 9 month, atleast 12 months or longer). (Although, as indicated above, in aspects ofthe invention which are not limited to administration for at least 12weeks, shorter administration periods may be possible, at least oneweek, at least two weeks, at least one month, or at least two months).As noted above, in other embodiments the GnRH antagonist may beadministered for at least 1, 2, 3, 5, 6, 7, 8, 9, 10 or 20 years orlonger. In some embodiments, short acting or long acting formulationsare utilized.

The GnRH antagonist may be administered by any convenient or desiredroute, including both parenterally or enterally. In some embodiments,the GnRH antagonist is administered subcutaneously, intramuscularly,intravenously, dermally, orally, infusion pump, or intraarticularly.Administration may be for example by injection or infusion or by othermeans of local or systemic delivery. In some embodiments, GnRHantagonists are administered up to 6 times daily or weekly or every 2-4week intervals, or monthly intervals or every 2-6 month intervals, oryearly, 0.1 mg to 3000 mg, e.g. 10 mg to 1000 mg. In some embodimentswith an initial loading dose between 20 mg to 1000 mg.

In some embodiments, a long acting GnRH antagonist, e.g degarelix,ozarelix or abarelix is administered weekly, or every 2-4 weekintervals, or monthly intervals or every 2-6 month intervals, or yearly,10 mg to 1000 mg. In some embodiments, with an initial loading dose ofthe long acting GnRH antagonist between 20 mg to 1000 mg.

In a particular embodiment a long-acting antagonist (which may be along-acting peptide antagonist, e.g. degarelix, or a sustained releasepreparation of a short-acting GnRH antagonist such as ASP1707) isadministered at an initial loading dose of 20 to 100 mg, followed by amaintenance dose of 40-1000 mg every 10-14 days, e.g. every 2 weeks. Forexample, the maintenance dose may be 60-1000, 80-1000, 100-1000, 60-800,60-500, 80-800, 80-500, 100-800, 100-500, 80-400, 80-320, 60-400,60-320, 60-160, 80-160, 60-150, 80-150 mg or any range between any ofthe above-mentioned integers.

In another embodiment, maintenance dose may be administered every 5 to10 days, e.g. every 7 days or every week. The maintenance dose may be inthe range as indicated above or may be 30-1000, 30-800, 30-500, 30-400,30-320, 30-300, 30-200, 30-150 mg, or it may be 40 or 50 mg to any oneof 1000, 800, 500, 400, 320, 300, 250, 200, 180, 160 or 150 mg.

In some embodiments, short acting GnRH antagonists, e.g. peptideantagonists such as cetrorelix or ganirelix are administered up to 6times daily (e.g. 2, 3 or 4 to 6 times daily) or daily or 2-6 timesweekly or weekly or every 2-4 weeks, e.g. at a dose of 0.1 mg to 30 mg.In particular, the antagonist may be administered at a dose of any oneof 0.1, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg to any one of 20,30, 40, 50, 60, 70, 80, 90 or 100 mg.

In some embodiments, an oral GnRH antagonist such as non-peptide oralGnRH antagonists, spiroindoline derivatives as GnRH antagonists,relugolix, elagolix, or ASP1707 is administered up to 6 times daily(e.g. 2, 3, 4 5 or 6 times daily), or daily, or 2-6 times weekly orweekly, e.g. at a dose of 0.1 mg to 3 g.

For example, elagolix may be administered at a dose of 10 to 2000mg/day, e.g. 200 to 800, 200 to 600, 200 to 500, 300 to 800, 300 to 600,300 to 500, 550 to 750, 750 to 1000 mg/day, e.g. 400 or 500 mg/day.

Relugolix may be administered at a dose of 10 to 1000 mg/day, e.g.20-100, 50-200, 100-500, 500-1000 mg/day, e.g. 400 mg/day.

ASP1707 may be administered at a dose of 0.5-200 mg/day, e.g. 1 to 150,1 to 100, 1 to 90, 1 to 80, 1 to 70 1 to 60, 1 to 50, 1 to 40, 1 to 30,1 to 20, 1 to 15, 1 to 10, 5 to 200, 5 to 150, 5 to 100, 5 to 90, 5 to80, 5 to 70, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 20, 5 to 10, 10 to200, 10 to 150, 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to50, 10 to 40, 10 to 30, 10 to 20, 20 to 200, 20 to 150, 20 to 100, 20 to90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, 50 to200, 50 to 150, 50 to 100, 50 to 90, 50 to 80, 50 to 70 or 50 to 60mg/day. In particular ASP1707 may be administered at a dose of 200mg/day, 150 mg/day, 100 mg/day, 90 mg/day, 80 mg/day, 70 mg/day, 60mg/day, 50 mg/day, 40 mg/day, 20 mg/day, 10 mg/day or 5 mg/day. In aparticularly preferred embodiment, ASP1707 is administered to thesubject twice a day, each dose being in the range from 5 to 50 mg.Preferably the doses are equal, and most preferably each dose is 30 mg(a total dose of 60 mg/day). A twice daily dosage of 5 mg, 10, mg, 15mg, 20 mg, 30 mg, 45 mg or 50 mg ASP1707 is particularly preferred.

A spiroindoline derivative may be administered at a dose of 0.1 to 1000mg/day, e.g. 0.1 to 10, 0.5 to 150, 150 to 500, 550 to 750 e.g. 200mg/day.

In general, we propose that a GnRH antagonist may be administered at ahigher dose than is presently proposed for uses of GnRH antagonists inthe art, particularly where such antagonists are used for treatinghormone-related problems, e.g. prostate cancer, fibroids orendometriosis, or for fertility treatment. For example such doses orconcentrations may be 3 or 4 times higher than typical, conventional ornormal doses or concentrations, e.g. doses or concentrations for suchtreatments.

As noted above, the GnRH antagonists are proposed according to theinvention described above for administration on a long-term basis, thatis for at least 12 weeks. However in a separate and alternative aspectalso included according to the invention is the administration of a GnRHantagonist at the doses indicated above, but without limitation to anadministration period of at least 12 weeks.

In some embodiments, ‘healthy’ people or ‘unhealthy’ people areidentified with increased levels of peripheral GnRH using ELISA GnRHdetection kits, PET scans for the detection of GnRH activity, tissuestaining, or otherwise i.e. screened, diagnosed, or prognosticallyevaluated for age associated inflammation, or chronic inflammation orinflammatory disease, with the aim of possible treatment initiation.Some of these people may fulfill criteria for diagnosis of aninflammatory disease or show signs or symptoms of chronic inflammationand some may not. These people can be, but are not limited to, otherwisehealthy people with normal blood pressure, normal cholesterol and normalhigh sensitivity CRP, or other. These people may also be people who havecardiovascular disease or osteoporosis. If the presence of peripheralGnRH has been identified, for example between 30 pg/mL to 1500 pg/mL, orbetween 20 pg/ml to 5000 mg/ml, or through another method, these peoplemay be offered a drug to lower the effects of peripheral GnRH. In someembodiments, the detrimental effects of peripherally detectable GnRH areexpected to decrease with treatment which decreases the effects of GnRHover time. It may be necessary to individually titrate levels of altereddownstream hormones, for example oestrogen or testosterone.

In some embodiments, the detection of peripheral GnRH will be positivelyassociated with the amount of systemic inflammation or the rate ofincrease of systemic inflammation. This is a method for the prognosticevaluation for disease(s) caused by inflammation.

GnRH is thought to be only detectable in the brain. GnRH is rapidlydegraded after reaching the pituitary. It has been suggested that theisolated hypophyseal portal system may not only have evolved solely as ameans to deliver hypothalamic peptides such as GnRH to the pituitary,but also as a way to prevent their delivery to extra-pituitary targetsresulting in unfavorable outcomes, such as systemic inflammation or ageassociated inflammation. Through the detection of GnRH activity outsidethe brain, people can be screened, diagnosed, evaluated, and treated forage-related inflammation, chronic inflammation or inflammatory diseases.

Thus, the present invention in a further aspect provides a GnRHantagonist for use in the treatment or prevention of peripheralinflammation, or systemic inflammation, or chronic low levelinflammation (particularly chronic systemic low level inflammation),including age-related inflammation (e.g. low level age-relatedinflammation).

In one embodiment, the invention provides a GnRH antagonist for use inthe treatment or prevention of inflammatory peripheral GnRH. Moreparticularly, the subject has a peripheral GnRH level of 160 pg/ml ormore, e.g. as measured in plasma or serum. In other embodiments, thesubject may have a peripheral GnRH level of at least 120% of theconcentration of healthy control subject(s), 100, 200, 320, 350, 370,380, 400, 420, 450, 470, 480 or 500 pg/ml or more.

In such aspects and embodiments the GnRH antagonist is preferablyadministered long term for at least 12 weeks.

Peripheral GnRH may be measured or determined in a sample of any bodytissue or fluid taken from the subject at or from a site or locationoutside of the brain (i.e. from the periphery of the body).Conveniently, the GnRH may be determined in a blood or a blood-derivedsample, particularly serum or plasma, but this can be in any otherblood-derived preparation. Other representative samples include vasculartissue and synovial fluid.

The GnRH may be determined or measured by any method or technique knownin the art. Conveniently an immunoassay may be used. More particularly,an immunoassay may be performed using an antibody (or antibody fragmentor antibody derivative etc.) which binds specifically to GnRH, and thebinding of the antibody to GnRH is detected or determined, e.g. anELISA, and kits for performing such assays are commercially available.Alternatively, GnRH may be detected with histochemical and/orhistological techniques e.g. immunohistochemical techniques.

Alternatively, peripheral GnRH may be determined by assays or methodscarried out in in vivo on the body, e.g. by imaging techniques. Thus forexample a PET scan may be performed for GnRH activity, e.g. using a GnRHantagonist radiolabelled with positron emitting nuclides for visualisingGnRH receptors.

In particular embodiments of these aspects, the subject may be a subjectwho does not have or exhibit any signs or symptoms of an inflammatorydisease. Accordingly, in particular embodiments the subject may have asub-clinical inflammatory condition, or may be without overt symptoms ofdisease, or overt clinical symptoms of disease, or may be healthy. Thus,as noted above, the subject may have normal blood pressure, and/ornormal cholesterol, and/or normal high sensitivity CRP, and/or normalblood lipids (e.g. triglycerides) etc. The subject may have a normalweight, or normal BMI parameters. In other embodiments the subject maybe overweight or obese or have increased BMI parameters etc. In otherembodiments, the subject may, as noted above, have low levelinflammation, or chronic systemic inflammation, or age-relatedinflammation.

The treatment of inflammatory peripheral GnRH, or indeed anyinflammatory condition, according to this aspect of the invention mayinvolve a combination therapy as discussed above, and in particular acombination therapy with a sex hormone or other agent effective forhormone substitution therapy, and in particular the sex hormone or otheragent may be titrated to baseline level or higher, as described above.

A method of the invention may accordingly involve determining a level ofperipheral GnRH in a subject, and if said level is higher than that of ayoung healthy adult or 160 pg/ml or above, administering a GnRHantagonist.

The step of determining the peripheral GnRH level may involve monitoringthe peripheral GnRH level in a subject over a period of time, e.g. over1, 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, 20 weeks or more, for example over1, 2, 3, 4, 5, 6, 7, 8, 9 months or more, or 1, 2 or 3 years or more.

Accordingly, in some embodiments of the invention, the GnRH antagonistmay be administered to a subject in whom the level of peripheral GnRHhas been determined, in particular pre-determined, and in particular ina subject who has been monitored for peripheral GnRH levels. The uses ofthe invention may thus combine a step of determining or screening forperipheral GnRH levels and a step of administering a GnRH antagonist ifthe peripheral GnRH level exceeds a particular threshold or cut-offvalue (e.g. of 160 pg/ml or 1600 pg/ml).

In another aspect, the invention provides a method for detecting ordetermining an inflammatory condition in a subject, said methodcomprising determining the level of peripheral GnRH in said subject. Inparticular such a method may, as discussed above, be performed on asample of a body tissue or fluid e.g. serum or plasma. In otherembodiments, as discussed above, an in vivo method, e.g. imaging orscanning may be used. In a particular embodiment the presence of aninflammatory condition is detected or determined if said level ofperipheral GnRH is 160 pg/ml or more. In a particular embodiment, thesubject may have low-level inflammation, or chronic systemicinflammation, or age-related inflammation. In other embodiments, thesubject may be healthy or may have no signs or symptoms of aninflammatory disorder, as discussed above.

In a further aspect, the invention also provides an agent capable ofdisclosing GnRH level and/or activity for detecting or determining invivo an inflammatory condition in a subject, Accordingly, in thisaspect, the invention may provide a diagnostic method or use practicedon the body of the subject, i.e. agent for use in diagnosing aninflammatory condition in a subject. Such an agent may be a GnRHantagonist, or a molecule capable of binding to the GnRH receptor or toGnRH (e.g. an affinity binding partner for GnRH or for the GnRHreceptor, such as antibody or fragment or derivative thereof), whichantagonist or molecule is provided with a label, particularly adetectable label, e.g. a radiolabel or positron-emitting nuclide or someother signal-giving label.

EXAMPLES

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

Example 1 Examples of Treating Patients with Rheumatoid Arthritis with aGnRH Antagonist Baseline Demographics

Variable Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Age(years) 56 58 45 71 78 71 Sex Female Female Premenopausal Male FemaleFemale Female Disease 10 26 17 16 31 38 Duration (years) Diseaseactivity ¹ 6.2 7.5 7.6 8.6   6.4 7.2 Number of 6 7 5 3    1 ² 5biologics previously failed Onset? Menopause Postpartum Postpartum Acuteonset at Menopause Postpartum the time of ear infection Duration/Effect4 infusions 4 months. 1 infusion 1 infusion 2 infusions 2 months. oflast biologic Rituximab TNF-inhibitor TNF-inhibitor Rituximab RituximabRoactemra (Mabthera) (Simponi/golilimumab) (Remsima) (Mabthera)(Mabthera) (Tocilizumab) (each 6 Increasing disease Side effects.Increasing 6 months apart. Very low white months apart). activityIncreasing disease disease Malignant cell count. No effect. documented.activity activity melanoma Increased documented. documented. diagnosis.disease activity Unknown effect documented. on disease activity. ¹Measured by DAS-28. >5.1 defined as high ² Patient cannot use biologics(other than rituximab), due to malignant melanoma.

Baseline and Final Visit Clinical Variables

Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Treatmentduration 28    15    12    9  7  36    (weeks) Degarelix loading dose,160 mg, Every 180 mg, Every 4 240 mg, Every 3½ 240 mg, Every 3 ½ 120 mg,Every 5 240 mg, Every intervals, maintenance 4 weeks to 3 ½ weeks to 3 ½weeks, 80 mg weeks, 80 mg weeks to 3 ½ 3 ½ weeks for doses weeks, 80 mgweeks, 80 mg stepped up 120 mg stepped up to 160 weeks, 80 mg 2 months,mg initially, then 80 mg every 2 weeks ACR 90% 60% 50% 50% 30% 80% %Response Swollen joint count 6, 0 17, 2  23, 4  24, 3  10, 2  16, 0assessed with ultrasound; baseline, final 9, 0 17, 3  26, 11 28, 11 12,0  24, 0 Tender joint; baseline, final Patient own assessment 81, 0  90,35 50, 30 90, 50  88, 88³ 60, 7 (0-100); baseline, final Physicianassessment 85, 7  90, 40 90, 30 80, 40 70, 20 90, 7 (0-100); baseline,final Function score (Good <1); 1.25, 0    2.0, 0.25 1.75, 0.63 0.88,0.38 1.29, 0.88   1.5, 0.625 baseline, final Morning stiffness (hrs);2.5, 0   9, 1  7, ½ 7, 3 2.5, ¾   2, 0 baseline, final ³Muscular pain inarms

Baseline and Final Visit Laboratory Variables

Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Laboratoryvariable By 24 wks By 15 wks By 12 wks By 9 wks By 7 wks By 3 ½ wks(reference range)⁴ Baseline; Final Baseline; Final Baseline; FinalBaseline; Final Baseline; Final Baseline; Final ESR (varies by age/sex)  46, 24 *  51, 16*   46, 20 * 78, 36 28, 8   37, 24* CRP (<5) 63, 1644, 23 33, 19 142, 56  <5, 6    22, <5* CK (35-210) 51, 61  25, 39*  32,55*  18, 43* 76, 61 74, 58 Hb (varies by sex) 12.2, 12.8 13.0, 13.311.4, 11.2 12.3, 12.8 11.4, 11.6 12.4, 13.2 LH (varies by age/sex)  22,<0.   36, <0.3   2, >0.3   8, <0.3    44, <0.3    30, <0.3 FSH (variesby age/sex) 38, 1   72, 0.4 6, 1   15, 0.7*    64, <0.1  79, 0.6Homocysteine (5-17) 10, 9  12, 13 31, 21 10, 10 10, 10 10, — Rheumatoidfactor (<14) 7, 9 51, 38 178, 163 >200, >200    167, >200 8, 8 Abovelimit CCP antibody (<10) NEG, NEG 101, 70  102, 101 >340, >340 257, 235NEG, NEG Above limit HbA1c (4.3-6.1)  6.4, 6.1* 5.3, 5.3 5.1, 5.4 6.9,6.4 5.1, 5.5 5.5, 5.4 ACTH (<10.2) 10.7, 6.1*   2.3, <1.1 3.9, 2.1  8.0, <1.1 11.3, 10.8 <1.1, <1.1 Blood Pressure   151/90, 120/71 *118/82, 126/80 110/68; 110/66 130/80; 120/78 134/79; 124/80  90/60;100/64 ⁴Italics denotes over reference range values, bold denotes underreference range values. ESR, erythrocyte sedimentation rate; CRP,C-reactive protein; CK, creatinine kinase; Hb, Hemoglobin; LH,luteinizing hormone; FSH, follicle-stimulating hormone; CCP, cycliccitrullinated peptide; HbA1c, glycated hemoglobin: ACTH,adrenocorticotrophic hormone

Other Variables Including Safety Parameters

Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Laboratoryvariable By 24 wks By 15 wks By 12 wks By 9 wks By 7 wks By 3 1/2 wks(reference range) Baseline; Final Baseline; Final Baseline; FinalBaseline; Final Baseline; Final Baseline; Final ALAT (10-45) 22, 26 14,21 16, 23 52, 37 65, 52 18, 22 Creatinine (varies with 56, 47 59, 59 42, 49* 49, 56 20, 29 61, 46 age/sex) LDL (2.0-5.4) 3.6, 3.8 2.8, 2.92.7, 2.3 2.7, 2.3 3.2, 3.2 2.2, 1.9 HDL (1.0, 2.7) 2.33, 2.31 1.93, 2.011.78, 1.42 1.78, 1.62 1.83, 1.64 1.71, 1.65 Cholesterol (3.9-7.8) 6.0,6.3 5.0, 5.6 4.8, 4.5 4.8, 4.5 5.2, 5.4 4.3, 4.1 Triglycerides(0.45-2.6) 1.19, 1.38 1.16, 1.39 1,23, 1.38 1.23, 1.38 0.93, 1.06 1.21,1.31 P1NP (varies with sex) 42, 43 33, 23 40, 38 39, 38 46, 35 — IGF-1(varies with age) 14.6, 12.0 19.1, 22.2 24.6, 25.9 24.6, 25.9 15.3, 12.0— Osteocalcin (varies with 2.2, 3.8 4.7, 3.4 2.7, 3.3 3.3, 2.7 4.9, 4.4sex) CTX-1 (varies with age 0.42, 0.36 0.37, 0.31 0.65, 0.71 0.68, 0.830.48, 0.49 — and sex) Apo (A1) 2.2, 2.2 2.0, 2.0 — — — — Apo (B) 1.1,1.2 0.9, 0.9 — — — — *Denotes normalized values

The figures show differences before and after treatment with degarelix.Patients have not increased their concomitant stable medications, andthey have not intraarticular, intramuscular or intravenous cortisonetherapy.

FIGS. 1A, 1B and 1C show images of arthritis in the foot of patient 1before and during treatment with degarelix. Patient 2 has returned toworking part-time and spinning hours at her local gym. She started 100%on sick benefits 3 years ago. Patient 3 used crutches to come to thebaseline consultation. She no longer requires crutches.

FIG. 2 shows ultrasound pictures of patient 5. FIG. 2A shows white areahighlighted by arrow, which is power Doppler denoting inflammatoryactivity in shoulder. FIG. 2B shows no power Doppler in same shoulder.FIG. 2C shows white area highlighted by arrow, which is power Dopplerdenoting inflammatory activity in right MCP 4 (finger joint). FIG. 2Dshows no power Doppler in right MCP4 joint. FIG. 2E shows white areahighlighted by arrow, which is power Doppler denoting inflammatoryactivity in left MCP 4 (finger joint). FIG. 2F shows no power Doppler inMCP 4 left side, and decreased joint fluid (oval). FIG. 3 shows patient6 before (FIG. 3A) and after (FIG. 3B) degarelix treatment.

FIG. 4 shows ultrasounds of patient 6. The first ultrasound (left) showsa large effusion (swelling indicated by black area) over the rightwrist. The second ultrasound (right) shows the same area by 3.5 weeks ofdegarelix treatment. The effusion on the right is much smaller, and nolonger painful. She halved her long term stable prednisolone dose from10 mg to 5 mg within days of the first degarelix injection. She hasreduced two shoe sizes and also halved her morphine and diuretic intake.

FIGS. 5A-F show disease activity variables of patient 2 whilst beingtreated with degarelix.

Example 2 Examples of Patients with Continued RA Improvement OverSeveral Weeks Despite Stably Low Levels of LH

Patient 3

Baseline 4 wks 8 wks 12 wks ACR % — 30% 40% 50% response LH 2 <0.3 <0.3<0.3 ESR 46 41 36 20

Patient 4

Baseline 3½ wks 9 wks ACR % — 20% 50% response LH 8 <0.3 <0.3 ESR 78 6836

Example 3 Example of Treating a Patient with Lupus with a GnRHAntagonist Social History

A male, 63 years old, married with 3 grown up children has been on sickbenefits since 2010 primarily due to lupus diagnosed in 1988.

History of Lupus

The patient has a medical history of a multimorbid patient. Systemiclupus erythematosus (SLE) was diagnosed in 1988 based on Raynauds, skinchanges, arthralgia, high titre lupus anticoagulant, ANA, SSA, and SSB.Chilblain type changes in hands and feet (FIGS. 6-8). He has previouslytried methotrexate, azathioprine, and mycophenolate mofetil, which werenot effective in reducing the pain in his extremities, and gaveintolerable side effects such as mouth ulcers. He has had a pacemakersince 1986, AV block II. The patient suffered cardiac arrest in 2012,coronary stenosis was observed. The patient had kidney failure since2012, required dialysis, stabilized creatinine around 150, osteoporosis,COPD, and is a smoker.

The patient has had severe pain in his hands and feet for the last 10years, especially under the soles of his feet. Has had continuous smallulcerations on his toes and hands, and pigmented skin. This pain wasdebilitating, and reduced his daily function. He walked with difficulty,and had atrophic musculature in both his lower extremities. He hadataxia in his lower extremities, especially left side, and minimalpatellar and Achilles reflexes. He was unbalanced, experienced numbness,and parasthesias, all documented in his medical journal from theneurology outpatients clinic April 2014. His electromyography findingsfrom 2010 support a diagnosis of thin fiber neuropathy related to lupus.Due to his painful condition, he has been followed up by a pain clinic.He was offered a tricyclic antidepressant, amitriptyline 25 mg in March2014 for his painful condition. This had no effect on his pain. In April2014 the amitriptyline was increased to 50 mg with no effect. This wassoon after increased to 150 mg daily with no effect. Thereafter he triedgabapentin 100 mg×3 up to 300 mg×3 up to 1200 mg×3 with no effect. Hehas also tried oxycodone and naloxone 5/2.5 mg but this increased thepain. He has tried pregabalin up to 150 mg×2 with no effect.

By September 2014, we had no treatment options left. Therefore, wedecided to offer him degarelix. GnRH antagonists have shownanti-inflammatory effects and therefore we considered that degarelix, adepot GnRH antagonist, may have a therapeutic effect in lupus. 09.15.14,he received his loading dose of degarelix, 240 mg s.c.

Assessment of Response

Variable (ref. range) 15 Sep. 2014 23 Sep. 2014 14 Oct. 2014 7 Nov. 2014FSH (<12) 14↑ 2 <0.1 <0.1 LH (<12) 11   <0.3 — <0.3 ANA (<1) >32↑ out 31 ↑  28↑  26↑ of range (25 Sep. 2014) Total urinary 0.78↑ 0.75 ↑  0.15 ²    0.17 protein g/L (++on urine (++on urine sticks) sticks)

Proteinuria decline is linked to improved lupus nephritis outcomes.Korbet et al studied 61 patients in a controlled trial comparingstandard therapy with plasmapheresis for severe lupus nephritis. Ofthese, 34 patients attained a 50% or greater reduction at 6 months and27 did not. The patient described in this case report obtained acomplete remission of proteinuria by 1 month, which continues to be inremission at 2 months. He had constant, increasing proteinuria in 2014.

By day 10 of treatment, several ulcers had healed (FIGS. 6B, 6D, 7B, and8), and the patient noticed a remarkable improvement. By day 5 hismorning pain had decreased from 3.5 hours to 1.5 hours; by day 10 to 0hours. This continues to be at 0 hours by 8 weeks. The decreasederythema at week 8 can be seen in FIG. 8. He told his physician he wasable to walk without shoes, something he had not been able to do forseveral years.

By 1 month of treatment, the patient took a holiday in Poland, and wasable to walk for several hours each day with the group he travelledwith. He no longer has parasthesias and his balance has improved. Hereceived his second dose of degarelix in October 2014, 80 mg s.c. Hecontinues on 80 mg s.c. every two weeks.

This example demonstrates that degarelix reduced several symptoms oflupus and increased quality of life in a lupus patient.

Example 4 Example of Treating a Patient with Ankylosing Spondylitis witha GnRH Antagonist Social History

73 year old male, married. 2 grown up children. Previously worked as awelder. Prior to retirement, he was on 100% sick benefits since 1993;50% since 1986.

History of Ankylosing Spondylitis

Diagnosed with ankylosing spondylitis in 1984, HLAB27 positive, withrepeated iridocyclitis. Used non-steroidal anti-inflammatory drugspreviously. He was offered the TNFα inhibitor Golilimumab in 2010. Hisdisease activity score was 8.9 in October 2010 (see below forexplanation of disease activity score) on initiating Golilimumab; by theJune 2012 this score had reduced to 6.6, but by May 2013 the effect hadworn off and his score was 7.6. May 2013, he initiated Enbrel treatment,but his disease activity increased from 7.6 to 8.3. Due to his poorresponse to TNFα therapy, he was offered the GnRH antagonist degarelixin September 2014.

Assessment of Response

Disease activity in ankylosing spondylitis is measured by the BASDAI(Bath Ankylosing Spondylitis Disease Activity Index (1)), which has beenextensively validated in clinical trials, 2, 3 and is one of the mostcommonly used outcome measures in clinical trials. This is a compositeindex that evaluates fatigue, axial and peripheral pain, stiffness andenthesopathy. His BASDAI was at 8.7 at baseline. In September 2014 hereceived his first dose of degarelix 240 mg s.c. By 2 weeks, his BASDAIhad reduced to 6.8, and by 6 weeks it had reduced to 5.8. This is thelowest BASDAI score he has ever had since we first started using thismethod of assessment 4 years ago in 2010. His morning stiffness lasted 4hours at baseline, by 6 weeks his morning stiffness had reduced to 2hours. He had his second dose of degarelix 80 mg s.c in October 2014.Lumbar flexion and maximum arm abduction were increased after 6 weeks oftreatment.

Response to therapy in clinical practice is defined as improvement of 2units or 50% on a 0-10 scale of the BASDAI, and expert opinion that thetreatment should be continued. His reduction by 6 weeks was 2.9 units,and 7.1 units with an 82% BASDAI score improvement by 9 weeks, and weshall continue treatment. A BASDAI 50% improvement is defined as a majorclinical response. He has also achieved an ASAS 80 (AnkylosingSpondylitis Assessment Group of at least 80% improvement) response. Forcomparison, approximately 50-60% of patients achieve a BASDAI 50 by 12weeks on TNF inhibitors (4). His ESR has decreased from 46 mm to 23 mm,which is his lowest ESR reading in 19 years. His serum urate hasdecreased from 515 to 426 μmol/L, which is also his lowest urate levelin 19 years. By 9 weeks of GnRH antagonist therapy, his kidney functionhas normalized, and fasting glucose reduced from 7.4 to 6.5 mmol/L. By 3months, the effect on his ankylosing spondylitis remained, creatininewas at 98 μmol/L, with a glomerular filtration rate of 66 ml/min/1.73 m²in December 2014.

Lab. Variable (Ref range.) 10 Sep. 2014 24 Sep. 2014 27 Oct. 2014 10Nov. 2014 ESR <20 mm  46↑  31↑  28↑   23↑ CRP <5  <5  <5  <5 Creatinine112↑ 114↑ 111↑ 99 (60-105 μmol/L) Glomerular  56↓  55↓  56↓ 65filtration rate (>60 ml/min/1.73 m²) Urate 515↑ 512↑ 495↑ 426  (230-480μmol/L) Glucose fasting  7.4 — —   6.5 (4.0-6.0 mmol/L)

Past Medical History

1952 Operated as an 11 year old with a left hip replacement due toepiphysiolysis as a child.1974 Cholecystectomy (gallbladder removal)1982 Myocardial infarction, underwent coronary angiography whereocclusion of the left anterior descending artery was shown.1984 Diagnosed with ankylosing spondylitis, with repeated bouts ofiridocyclitis in association with ankylosing spondylitis1998 Left hip replacement for second time, right hip replacement; deepvenous thrombosis2009 Colitis, biopsy confirmed in 20102009 Carpal tunnel syndrome surgery in right hand; same in left hand20102014 MGUS with M-component at 2.4 g/L2014 Mild kidney failure

REFERENCES FOR EXAMPLE 4

-   1. Garrett et al. A new approach to defining disease status in    ankylosing spondylitis: The Bath Ankylosing Spondylitis Disease    Activity Index (BASDAI) J Rheumatol 1994; 1: 2286-91.-   2. Haywood et al. Patient-assessed health in ankylosing spondylitis:    a structured review. Rheumatology 2005; 44:577-86.-   3. Calin et al. Defining disease activity in akylosing spondylitis:    is a combination of variables (Bath Ankylosing Spondylitis Disease    Activity Index) an appropriate instrument? Rheumatology 1999;    38:878-82.-   4. Rudwaleit M, et al. Prediction of a major clinical response    (BASDAI 50) to tumour necrosis factor alpha blockers in ankylosing    spondylitis. Ann Rheum Dis 2004; 63:665-670.

Example 5 Example Improving Bone Mineral Density in Patients Taking aGnRH Antagonist

Bone mineral density was measured in patients 1 and 2 (See Example 1 forpatient information). FIG. 9 shows that patient 1 shows an improvementin her bone mineral density in her hips for the first time in 3.5 years.She was not taking any therapies for osteoporosis during this period.This was unexpected as the summary of product characteristics statesthat a decrease in bone mineral density is expected under GnRHantagonist treatment. However, our data indicates the opposite. Despitehaving low oestrogen, using concomitant prednisolone and longstandingrheumatoid arthritis (all of which are independent risk factors forosteoporosis), her bone mineral density improved during GnRH antagonisttreatment.

FIG. 10 shows an improvement in her bone mineral density in the hips ofpatient 2 for the first time in 4.5 years. This was unexpected as thesummary of product characteristics states that a decrease in bonemineral density is expected under GnRH antagonist treatment. Despiteusing having low oestrogen, using concomitant prednisolone andlongstanding rheumatoid arthritis (all of which are independent riskfactors for osteoporosis), her bone mineral density improved during GnRHantagonist treatment.

Example 6 Example of Reducing Cardio-Vascular Disease Risk in PatientsTaking a GnRH Antagonist

The summary of product characteristics for degarelix states thefollowing under section

‘4.4 Special Warnings and Precautions for Use’:

-   -   Development or aggravation of diabetes may occur, therefore        diabetic patients may require more frequent monitoring of blood        glucose (and therefore HbA1c) when receiving androgen        deprivation therapy.    -   Cardiovascular disease such as stroke and myocardial infarction        has been reported in the medical literature in patients with        androgen deprivation therapy. Therefore, all cardiovascular risk        factors should be taken into account.        Therefore it is unexpected that degarelix could be beneficial in        cardiovascular disease. However, our data below indicates this.

In our patient group who were offered degarelix, some patients had riskfactors for cardiovascular disease, metabolic syndrome, lipidemia orhypoalphalipoproteinemia (low HDL). None of these patients had recentlystarted medications prior to the GnRH antagonist that could explain thechanges in blood pressure, HbA1c, fasting glucose or lipids. See Example1 for patient information.

Patient 1 with metabolic syndrome fulfilling 3 of 5 metabolic riskfactors (large waistline >35 inches, high blood pressure and highfasting glucose). After degarelix treatment, she no longer fulfills thecriteria for metabolic syndrome.

-   -   1. High HbA1c, contributes to CVD (normal reference range        4.3-6.1%) At baseline 6.4%, high. By 2 weeks HbA1c had        normalized to 6.1%. This continues to be normal, and at 8 months        is 5.7%. Her fasting glucose was 6.6 mmol/L in September 2014,        by October 2014 this was reduced to 6.3 mmol/L.    -   2. High blood pressure, contributes to CVD at baseline 151/90        (measured at several times prior to baseline with repeatedly        high blood pressure). Blood pressure by 6 months 120/71, and        this continues to be normal. At her last check up at 8 months,        her blood pressure was 118/78.    -   3. No other abnormalities at baseline for assessment

Patient 4

-   -   High HbA1c, contributes to CVD (normal reference range        4.3-6.1%). At baseline the HbA1c was 6.9%, high. By 1 month the        HbA1c had reduced to 6.6, by 2 months 6.4, and by 4 months 6.2,        and by 12 months it was normalized to 5.9. His hospital records        from a nearby hospital stated that his fasting glucose was high        at 8.5 mmol/L (reference range 4.0-6.0) in December 2013, and by        April 2014 this was higher at 8.9. They asked the patient's        family doctor to initiate treatment due to the development of        diabetes type II. His fasting glucose had reduced to 6.6 mmol/L        by 3 months after starting degarelix, and by 4 months has now        normalised to 6.0 mmol/L. No other abnormalities baseline for        assessment

Patient 7

-   -   1. Low HDL, contributes to CVD (normal reference range 1.0-2.7        mmol/L). Mora et al. investigated the link between cholesterol        and cardiovascular events in women and found baseline HDL level        was consistently and inversely associated with incident coronary        and CVD events. A low HDL cholesterol level is thought to        accelerate the development of atherosclerosis because of        impaired reverse cholesterol transport and possibly because of        the absence of other protective effects of HDL, such as        decreased oxidation of other lipoproteins.        -   She had low HDL at baseline 0.68 mmol/L in September 2014.            She received her loading dose of degarelix 240 mg s.c. in            September 2014. By 1 week, her HDL level had increased to            0.73, and by 2 weeks it continued to increase towards normal            at 0.75. By 3 weeks it had normalized to 1.13 mmol/L and at            4 weeks is normal at 1.19 mmol/L.    -   2. No other abnormalities at baseline for assessment

Patients 2, 3, 5, 6

These patients had no abnormalities (HbA1c, blood pressure, lipids) toindicate cardiovascular morbidity at baseline.

Example 7 Example of Treating a Patient with Inflammatory Bowel Diseasewith a GnRH Antagonist Patient History

-   -   73 year old male, diagnosed with colitis aged 69 yrs.    -   Inflammatory bowel disease, colitis verified on colonic biopsy        sample. Patients with inflammatory colitis usually have a higher        incidence of autoimmune diseases. This patient also had        ankylosing spondylitis.

Experimental Treatment

-   -   September 2014 received first dose of degarelix 240 mg s.c.    -   Received degarelix 80 mg s.c. approximately every two weeks        thereafter.

Assessment of Response

-   -   The inflammatory bowel diseases (IBDs), Crohn's disease and        ulcerative colitis, are chronic relapsing, remitting disorders.        The assessment of disease activity presents challenges to        clinicians. Fecal biomarkers, such as fecal calprotectin, are a        non-invasive method which can be used to aid managing        clinicians. Calprotectin is released extracellularly in times of        cell stress or damage and can be detected within feces and thus        be used as a sensitive marker of intestinal inflammation. Fecal        calprotectin has been shown to be useful in the diagnosis of        IBD, correlates with mucosal disease activity, and can help to        predict response to treatment or relapse. Fecal calprotectin        levels are elevated in patients with both Crohn's disease and        ulcerative colitis (1-2). There have been several studies        looking at the use of fecal calprotectin to predict or monitor        response to treatment.        -   In a study looking at 11 patients with relapsing IBD, fecal            calprotectin was analysed at inclusion and after 8 weeks of            treatment. Treatment was individualized medical therapy. A            normalized fecal calprotectin concentration at 8 weeks            predicted a complete response in 100% of patients (3),            corresponding to endoscopic mucosal healing (4). It has been            shown that in patients with steroid induced clinical            remission fecal calprotectin levels can remain elevated            (5-6). This finding is in keeping with earlier studies            showing incomplete mucosal healing in patients treated with            corticosteroids (7).        -   Fecal calprotectin concentration decreases significantly at            week 2 after an infliximab infusion (anti-TNFα) (8).

A level of <50 mg/kg is considered normal or remission for IBD.

This patient gave three fecal samples in total.

He started a GnRH antagonist 10.09.14.

3 samples in total 24 Sep. 2014 2 Oct. 2014 10 Jan. 2015 Fecalcalprotectin 79, high 48, normal 32, normal mg/kg (<50 normal)

-   -   There is no gold standard index in the measurement of colitis        disease activity in clinical trials. Due to the invasive nature        of endoscopies, the Mayo non-invasive colitis assessment of        response score can be used to assess treatment effect of IBD in        clinical trials (9-11). This score has been shown to correlate        well with the total Mayo score (12). Both the partial Mayo and        total Mayo score are used to assess disease activity in clinical        trials (13). The partial Mayo score also had good discriminatory        value between subjects in remission and those with active        disease (14). A clinical response has been defined as a 3 point        or greater change in the partial Mayo score (15).

This patient had a partial Mayo score of 3 at baseline, and 0 at 12weeks. A score of 0 is clinical remission. This is supported by his highfecal calprotectin score around baseline, reduced fecal calprotectinlevel at 4 weeks and further reduced calprotectin level by 12 weeks.

This example demonstrates GnRH antagonist treatment in a patient withcolitis improved his disease activity, as well as normalizing his fecalcalprotectin levels. As fecal calprotectin is high in IBD's in general,such as colitis and Crohn's disease, GnRH antagonists are beneficial inIBD's in general.

REFERENCES FOR EXAMPLE 7

-   ¹Røseth A G, et al. Assessment of disease activity in ulcerative    colitis by fecal calprotectin, a novel granulocyte marker protein.    Digestion 1997; 58: 176-180.-   ²Tibble J, et al. A simple method for assessing intestinal    inflammation in Crohn's disease. Gut 2000; 47: 506-513.-   ³Wagner M, et al. Fecal markers of inflammation used as surrogate    markers for treatment outcome in relapsing inflammatory bowel    disease. World J Gastroenterol. 2008; 14: 5584-89.-   ⁴Røseth A G, et al. Normalization of fecal calprotectin: a predictor    of mucosal healing in patients with inflammatory bowel disease.    Scand J Gastroenterol 2004; 39: 1017-1020.-   ⁵Sipponen T, et al. Fecal calprotectin and lactoferrin are reliable    surrogate markers of endoscopic response during Crohn's disease    treatment. Scand J Gastroenterol 2010; 45: 325-331.-   ⁶Kolho K L, et al. Fecal calprotectin remains high during    glucocorticoid therapy in children with inflammatory bowel disease.    Scand J Gastroenterol 2006; 41: 720-725.-   ⁷Modigliani R, et al. Clinical, biological, and endoscopic picture    of attacks of Crohn's disease. Evolution on prednisolone. Groupe    d'Etude Therapeutique des Affections Inflammatoires Digestives.    Gastroenterology 1990; 98: 811-818.-   ⁸Digestive Disease Week and the 107th Annual Meeting of the American    Gastroenterological Association Institute, May 20-25, 2006, Los    Angeles, Calif., USA. Abstracts. Gastroenterology 2006; 130: A1-911.-   ⁹Dhanda A D, et al. Can endoscopy be avoided in the assessment of    ulcerative colitis in clinical trials? Inflamm Bowel Dis 2012; 18:    2056-62.-   ¹⁰Higgins P D, et al. Patient defined dichotomous endpoints for    remission and clinical improvement in ulcerative colitis. Gut 2005;    54: 782-8.-   ¹¹Higgins P D, et al. Is endoscopy necessary for the measurement of    disease activity in ulcerative colitis? American Journal of    Gastroenterology 2005; 100: 355-61.-   ¹²Lewis J D, et al. Use of the noninvasive components of the Mayo    score to assess clinical response in ulcerative colitis. Inflamm    Bowel Dis 2008; 14: 1660-66.-   ¹³Rutgeerts P, et al. Infliximab for induction and maintenance    therapy for ulcerative colitis. N Engl J Med; 2005; 353: 2462-76.-   ¹⁴Turner D, et al. A systematic prospective comparison of    noninvasive disease activity indices in ulcerative colitis. Clin    Gastroenterol Hepatol 2009; 7: 1081-1088.-   ¹⁵Lewis J D, et al. Use of the noninvasive components of the Mayo    score to assess clinical response in ulcerative colitis. Inflamm    Bowel Dis 2008; 14: 1660-66.

Example 8 Example of Treating a Patient with Psoriasis with a GnRHAntagonist Patient History

-   -   38 year old male, diagnosed with lithium induced psoriasis aged        26 yrs.    -   His psoriasis was severe (erythema, induration and scaling),        with a baseline Psoriasis Area Severity Index (PASI (1)) score        of 53.6. The PASI score is a standardized score for assessing        disease activity in psoriasis.

Experimental Treatment

-   -   October 2014 received first dose of degarelix, 240 mg s.c.    -   He received degarelix 80 mg s.c. every two weeks thereafter.    -   His testosterone levels decreased from 12 nmol/L to <1 nmol/L        (normal 8-35 nmol/L) after the first week of GnRH antagonist        therapy. He therefore received testosterone supplementation in        the form of Testogel containing 50 mg of testosterone, first        daily; thereafter every 3 days to maintain testosterone levels        within the normal range. At week 8 his testosterone levels were        normalized at 9 nmol/L (under testosterone substitution and a        GnRH antagonist.

Assessment of Response

-   -   By 8 weeks his PASI score was 41.3; an absolute reduction of        12.3 points and a 23% reduction. This improvement in his        psoriasis is stable at 6 months.    -   High levels of serum uric acid in patients with psoriasis is a        result of increased purine catabolism due to rapid epidermal        cell turnover, and can be used as a marker of disease activity        in psoriasis (2). One study showed after 12 weeks of psoriasis        treatment, mean values of serum uric acid were found to be        significantly decreased in patients with psoriasis (3).    -   This patient had a serum uric acid (reference range 230-480 mon)        of 485 mon at baseline; and this was normalized by 8 weeks at a        level of 432 mon.    -   Lithium-induced psoriasis is particularly resistant to therapy.        There are very few trials of lithium-induced psoriasis. One        trial examined 15 patients with psoriasis who were taking        lithium. Their psoriasis was mild, with a median pretreatment        PASI score of 4.9. After 10 weeks of inositol therapy, their        median PASI score was reduced by 1.7 points, or by 35% (4).    -   The etiology of lithium-induced psoriasis is uncertain, but may        share some aspects with other types of psoriasis because lithium        has also been reported to exacerbate existing psoriasis (5). Due        to the effect on lithium-induced psoriasis, with the decrease in        serum uric acid levels which can be increased in psoriasis of        any type, it is expected that GnRH antagonists will have a        beneficial effect in psoriasis in general.

This example demonstrates improved signs and symptoms in a patient withpsoriasis treated with a GnRH antagonist treatment. This example alsodemonstrates the effective combination of a GnRH antagonist with gonadalhormone replacement, in this case testosterone replacement, in aninflammatory disease.

REFERENCES FOR EXAMPLE 8

-   ¹Fredsiksson T, et al. Severe psoriasis-oral therapy with a new    retinoid. Dermatologica 1978; 157: 238-44.-   ²Jain V K, et al. C-reactive protein and uric acid levels in    patients with psoriasis. Ind J Clin Biochem 2011; 26: 309-311.-   ³Jam V K, et al. C-reactive protein and uric acid levels in patients    with psoriasis. Ind J Clin Biochem 2011; 26: 309-311.-   ⁴Allan S J R, et al. The effect of inositol supplements on the    psoriasis of patients taking lithium: a randomized,    placebo-controlled trial. Br J Derm 2004; M 150: 966-969.-   ⁵Skoven I, Thompson J. Lithium compound treatment and psoriasis.    Arch Dermatol 1979; 115: 1185-7.

Example 9 Example of Treating a Patient with Systemic Sclerosis with aGnRH Antagonist Patient History

-   -   55 year old female, diagnosed with systemic sclerosis aged 52        yrs.    -   Fulfilled the 2013 EULAR/ACR classification criteria for        systemic sclerosis (1).    -   Diagnosis based upon Raynauds phenomenon, positive ANA, high        titre anti-Scl 70 antibodies, marked skin changes typical of        scleroderma, and esophageal dysmotility.    -   Previously tried cyclophosphamide and mycophenolate mofetil.    -   Progressive disease and worsening scleroderma skin features,        despite therapy.    -   Rapid progressive cardiac disease, verified by biopsy.    -   Experimental treatment    -   5 Dec. 2014 received first dose of degarelix, 240 mg s.c.    -   9 Jan. 2015 received second dose of degarelix, 80 mg s.c.

Assessment of Response at 6 Weeks

-   -   Digital ulcers and pitting scars are a characteristic feature of        systemic sclerosis, contributing to disability and pain. They        are important factors in patients' evaluations of quality of        life (2). Their precise objective evaluation can be included in        randomized controlled trials (3). Their pathogenesis is unclear.        Histologically, there is a plug-like hyperkeratosis with        parakeratosis, homogenized collagen fibers and perivascular        mononuclear cell infiltration (4). The improvement observed in        this patient (FIGS. 12 and 13) was unexpected, as trials do not        normally show a significant improvement in digital ulcers. For        example, a trial with cyclophosphamide showed that digital        ulcers increased on average from 7 to 9 ulcers, over a period of        12 months (5).    -   Other measures of response to systemic sclerosis treatment        include the Scleroderma Health Assessment Disease Activity Index        and the associated Visual Analogue Scales, as shown in the table        below.        Efficacy variables in a systemic sclerosis patient treated with        a GnRH antagonist at baseline, 5 weeks and 6 weeks:

Baseline 5 weeks 6 weeks 5 Dec. 2014 9 Jan. 2015 19 Jan. 2015 ModifiedRodnan 42 40 40 Skin Score Digital tip 5 2 2 ulcers/pitting scars HAQDI* 1.88 1.38 1.38 VAS** Pain 100 10 10 VAS Fingertip 100 40 30pain/pitting scars/ulcers VAS 100 40 30 Raynauds/vascular VAS Global 10040 40 VAS Lung/dyspnea 100 80 50 VAS Fatigue 100 75 60 VAS 100 80 60Gastrointestinal Symptoms Sleep 2 hrs/night 5 hrs/night 5.5 hrs/night*HAQ DI, Health Assessment Questionnaire Disease Index; **VAS, VisualAnalogue ScaleThis example demonstrates improved signs and symptoms in a patient withsystemic sclerosis treated with a GnRH antagonist.

REFERENCES FOR EXAMPLE 9

-   ¹Van den Hoogen, et al. 2013 Classification Criteria for systemic    sclerosis. Arthritis & Rheum 2013; 65: 2737-47.-   ²Malcarne V L, et al. Medical signs and symptoms associated with    disability pain and psychological adjustment in systemic sclerosis.    J Rheumatol 2007; 34: 359-67.-   ³Amanzi L, et al. Digital ulcers in scleroderma: staging    characteristics and sub-setting through observation of 1614 digital    lesions. Rheumatology 2010; 49:1374-82.-   ⁴Maeda M, et al. Pitting scars in progressive systemic sclerosis.    Dermatology 1993; 187: 104-8.-   ⁵Au K, et al. Course of dermal ulcers and musculoskeletal    involvement in systemic sclerosis patients in the scleroderma lung    study. Arthritis Care Res 2010; 62: 1772-8.-   ⁶Steen V D, et al. The value of the Health Assessment Questionnaire    and special patient-generated scales to demonstrate change in    systemic sclerosis patients over time. Arthritis Rheum 1997; 40:    1984-91

Example 10 Example of Treating a Patient with Rheumatoid Arthritis UsingCetrorelix Patient History

-   -   50 year old female with seropositive rheumatoid arthritis with        disease duration of 10 years.    -   Fulfilled ACR 2010 classification criteria for rheumatoid        arthritis.

Experimental Treatment

-   -   Had tried all therapies available previously without effect.    -   Offered experimental GnRH antagonist therapy, cetrorelix in 0.5        mg s.c. daily in October 2014, then 0.75 mg s.c. daily in        December 2014.

Assessment of Response at 12 Weeks

-   -   Disease activity score (DAS) reduction from 6.1 to 5.5.    -   A DAS reduction of 0.6 units is defined as a clinical response        to treatment.    -   Her ESR normalized from 35 mm to 28 mm, with a decrease in her        CRP from 41 mg/L to 18 mg/L. Her rheumatoid factor level has        also decreased from 137 kIU/L to 111 kIU/L.        This example demonstrates that cetrorelix, a GnRH antagonist,        improved signs and symptoms in a patient with rheumatoid        arthritis.

Example 11 Example of Treating a Patient with Spondyloarthritis with aGnRH Antagonist Patient History

-   -   38 year old male, diagnosed with spondyloarthritis aged 38 yrs.    -   Fulfilled the ASAS criteria for axial radiographic        spondyloarthritis.

Experimental Treatment

-   -   October 2014 received first dose of degarelix, 240 mg s.c.    -   Received degarelix 80 mg s.c. every two weeks thereafter.

Assessment of Response

Disease activity in ankylosing spondylitis/spondyloarthritis is measuredby the BASDAI (Bath Ankylosing Spondylitis Disease Activity Index (1)),has been extensively validated in clinical trials (2-3), and is one ofthe most commonly used outcome measures in clinical trials. This is acomposite index that evaluates fatigue, axial and peripheral pain,stiffness and enthesopathy. His BASDAI was at 3.8 at baseline. InOctober 2014 he received his first dose of degarelix 240 mg s.c. By 2weeks, his BASDAI had reduced to 2.9, and by 8 weeks it had reduced to1.3, remission. At 4 months, his BASDAI continues to be low at 1.5.

-   -   Response to therapy in clinical practice is defined as        improvement of 2 units or 50% on a 0-10 scale of the BASDAI, and        expert opinion that the treatment should be continued. His        reduction by 4 months was 2.3. We shall continue treatment.        This example demonstrates improved signs and symptoms in a        patient with spondyloarthritis treated with a GnRH antagonist.

REFERENCES FOR EXAMPLE 11

-   ¹Garrett et al. A new approach to defining disease status in    ankylosing spondylitis: The Bath Ankylosing Spondylitis Disease    Activity Index (BASDAI) J Rheumatol 1994; 1: 2286-91.-   ²Haywood et al. Patient-assessed health in ankylosing spondylitis: a    structured review. Rheumatology 2005; 44:577-86.-   ³Calin et al. Defining disease activity in akylosing spondylitis: is    a combination of variables (Bath Ankylosing Spondylitis Disease    Activity Index) an appropriate instrument? Rheumatology 1999;    38:878-82.

Example 12 Example of Treating a Patient with Multiple Sclerosis with aGnRH Antagonist Patient History

-   -   58 year old female, diagnosed with multiple sclerosis (MS) aged        50 years. Her MS was of the progressive type; characterized by a        steady deterioration in function.    -   Fulfilled the 2010 McDonald Criteria for the diagnosis of MS        (1).

Experimental Treatment

-   -   So far results of clinical trials for progressive MS have        generally been disappointing. Currently, there is no        FDA-approved treatment for progressive MS disease.    -   December 2014 received first dose of degarelix 240 mg s.c.    -   Received degarelix 80 mg s.c. every two weeks thereafter.

Difficulties in Assessing Response in Progressive MS

-   -   Almost all trials in progressive MS show a worsening in function        over time. The primary endpoint of these trials is usually to be        able to show less worsening in the active treatment group        compared to the placebo/other treatment group. Worsening is        often defined as an increase in the Expanded Disability Status        Scale (EDSS). EDSS is severely restrictive as an outcome measure        for trials in progressive sclerosis (2). Trials have not shown        improvements in the EDSS or function in MS patients (2). Optimal        outcome measures in progressive MS are open to debate.    -   Effective imaging outcomes in clinical trials have allowed for        the development of treatments for the relapsing-remitting type        of MS. Unfortunately, similar outcomes do not exist in        progressive MS. One of the most important factors in choosing        outcome measures for these patients is the meaningfulness to        patients.

Assessment of Response at 6 Weeks

General Function:

-   -   The patient reported a general improvement in her function, with        the ability to be able to use her MS affected lower limbs better        than she had been able to in the past 1 year, with continuing        gradual improvement under GnRH antagonist treatment. This was        particularly evident in her ability to raise her feet onto a        step, physiotherapy exercises, and dressing. In the past year,        she previously had always required her husband's help to climb a        step at home, or to carry out certain lower limb exercises with        her physiotherapist. By 4 weeks of GnRH antagonist treatment,        she no longer required her husband's/physiotherapist's help to        perform these activities. She climbed onto a step unaided at her        6 week check-up. The improvement started at around 4 weeks after        the initiation of therapy, and continued to improve further by 6        weeks.

Expanded Disability Status Scale (EDSS):

-   -   This patient's EDSS has remained stable at 6.5, as expected over        such a short time period of 6 weeks. A score of 6.5 is        equivalent to only being able to walk with the aid of a walker,        as in her case. This was evident at baseline and 6 weeks.

Multiple Sclerosis Impact Scale 29 (MSIS-29):

-   -   Patient-reported outcomes are of increasing importance in trials        of progressive multiple sclerosis (2). The most frequently used        global patient-reported outcome in multiple sclerosis is the        MSIS-29 (3). This has been correlated with clinical and imaging        metrics specifically in progressive forms of the disease (4).        According to a recent review (2), patient reported outcomes in        trials of progressive multiple sclerosis can be used to validate        MRI or clinical metrics, and increased use of patient-reported        outcomes in trials of progressive multiple sclerosis will be        expected to help satisfy regulators' requirements that        treatments show relevant benefit for patients.    -   At baseline, she scored 121 on the MSIS-29. By 6 weeks, her        score was reduced to 110. A clinically significant minimal        difference is a score change of 8. Therefore this reduction of        11 in her score is considered clinically significant (5).

Multiple Sclerosis Walking Scale-12 (MSWS-12)

-   -   The MSWS-12 (6) was designed as a disease-specific patient-based        instrument for use in clinical trials and clinical practice, to        capture the complex impact of MS on walking ability. The MSWS-12        has been extensively evaluated in MS, with demonstration of        internal consistency, high reliability and validity, and good        generalizability (7, 8). A strong correlation between the        MSWS-12 and accelerometer counts was observed (9), suggesting a        measurable relationship between objective mobility and a        patient's perception of his or her walking ability. One of the        most important attributes of the MSWS-12 in comparison to other        assessments of walking in MS patients is its responsiveness to        change. During validations, the MSWS-12 was shown to be more        responsive than other walking-based measures, including the EDSS        and the timed 25 foot walk (T25FW).    -   At baseline, she scored 60 on the MSWS-12. By 6 weeks, her score        was reduced to 53. This is a 12% improvement. A minimal        clinically significant difference has not yet been established.

Modified Fatigue Impact Scale for Multiple Sclerosis-5 Item Version(MFIS-5)

-   -   The MFIS-5 is a modified form of the Fatigue Impact Scale (10)        based on items derived from interviews with MS patients        concerning how fatigue impacts their lives. This instrument        provides an assessment of the effects of fatigue in terms of        physical, cognitive, and psychosocial functioning.    -   At baseline, she scored 20 on the MFIS-5. By 6 weeks, her score        was reduced to 12. This was a reduction of 40%, which is        clinically significant.

Timed 25 Foot Walk (T25FW)

-   -   The T25FW is another common endpoint in trials. At baseline, she        took 16.7 seconds to walk 25 feet, and by 6 weeks she took 15.2        seconds. This improvement in walking speed supports the        patient's reported improved outcomes.        The significant improvement in the MSIS-29 is supported by her        significantly improved fatigue in the MFIS-5, her improvement in        the MSWS-12, shorter T25FW time, and stable EDSS. This example        demonstrates improved signs and symptoms in a patient with MS        treated with a GnRH antagonist. This improvement in progressive        MS is likely to also be observed in the relapsing-remitting type        of MS. This is because the relapsing-remitting type gives        similar signs and symptoms as the progressive type to patients        over time.

REFERENCES FOR EXAMPLE 12

-   ¹Polman C H, et al. Dignostic criteria for multiple sclerosis: 2010    revisions to the McDonald Criteria. Ann Neurol 2011; 69: 292-302.-   ²Ontaneda D, et al. Clinical trials in progressive multiple    sclerosis: lessons learned and future perspectives. Lancet Neurol    2015; 14: 208-23.-   ³Hobart J, et al. The multiple sclerosis impact scale (MSIS-29): a    new patient-based outcome measure. Brain 2001; 124: 962-73.-   ⁴Hayton T, et al. Clinical and imaging correlates of the multiple    sclerosis impact scale in secondary progressive multiple sclerosis.    J Neurol 2012; 259: 237-45.-   ⁵Costellow L, et al. The patient knows best: Significant change in    the physical component of the Multiple Sclerosis Impact Scale    (MSIS-29 physical). J Neurol Neurosurg Psychiatry 2007; 78: 841-844.-   ⁶Hobart J C, et al. Measuring the impact of MS on walking ability:    the 12-Item MS Walking Scale (MSWS-12). Neurology 2003; 60: 31-36.-   ⁷Motl R W, et al. Confirmation and extension of the validity of the    Multiple Sclerosis Walking Scale-12 (MSWS-12). J Neurol Sci 2008;    268: 69-73.-   ⁸McGuigan C, et al. Confirming the validity and responsiveness of    the Multiple Sclerosis Walking Scale-12-   ⁹Motl R W, et al. Confirmation and extension of the validity of the    Multiple Sclerosis Walking Scale-12 (MSWS-12). J Neurol Sci 2008;    268: 69-73.-   ¹⁰Fisk J D, et al. Measuring the functional impact of fatigue:    initial validation of the fatigue impact scale. Clin Infect Dis 18    Suppl 1: S79-831994.    Examples 13 and 14 describe the treatment of RA using particularly    different dosing schedules.

Example 13 Treatment Using Degarelix 40 mg Weekly Patient History

-   -   57 year old female with seropositive rheumatoid arthritis with        disease duration of 22 years.    -   Fulfilled ACR 2010 classification criteria for rheumatoid        arthritis.

Experimental Treatment

-   -   Had tried most therapies available (for example: Humira,        Remicade, Enbrel, Actemra, several disease modifying        anti-rheumatic drugs), previously without effect or intolerable        side effects.    -   Offered experimental GnRH antagonist therapy, degarelix 40 mg        every week with 240 mg loading dose at baseline.

Assessment of Response

-   -   Disease activity score (DAS) reduction from 7.3 to 6.7 at 2        weeks.    -   A DAS reduction of 0.6 units is defined as a clinical response        to treatment (1).    -   Her ESR has decreased from 62 mm to 38 mm, with a decrease in        her CRP from 87 mg/L to 67 mg/L.    -   This example demonstrates that degarelix, a GnRH antagonist,        given as 240 mg loading dose with 40 mg weekly thereafter,        improved signs and symptoms in a patient with rheumatoid        arthritis.

REFERENCE FOR EXAMPLE 13

-   ¹Van Gestel A M, et al. Development and validation of the European    League Against Rheumatism response criteria for rheumatoid    arthritis. Arthritis Rheum 1996; 39: 34.40.

Example 14 Treatment Using Ganirelix Patient History

-   -   50 year old female with seropositive rheumatoid arthritis with        disease duration of 10 years.    -   Fulfilled ACR 2010 classification criteria for rheumatoid        arthritis.

Experimental Treatment

-   -   Had tried almost all available rheumatoid arthritis therapies        available previously without effect.    -   Offered experimental GnRH antagonist therapy, ganirelix 0.75 mg        s.c. daily (0.25 mg s.c. three times a day).

Assessment of Response at 4 Months

-   -   Disease activity score (DAS) reduction from 5.5 to 2.2        (remission).    -   A DAS reduction of 0.6 units is defined as a clinical response        to treatment (1).    -   Her ESR decreased from 28 mm to 19 mm, with a normalization of        her CRP from 23 mg/L to 5 mg/L. This is supported by the        decrease in her anti-cyclic citrullinated peptide (CCP)        antibodies from 27 μ/mL to 14 μ/mL. Her rheumatoid factor level        has also decreased from 113 kIU/L to 75 kIU/L.        This example demonstrates that ganirelix, a GnRH antagonist,        improved signs and symptoms in a patient with rheumatoid        arthritis.

REFERENCE FOR EXAMPLE 14

-   ¹Van Gestel A M, et al. Development and validation of the European    League Against Rheumatism response criteria for rheumatoid    arthritis. Arthritis Rheum 1996; 39: 34.40.

Example 15 Example of Treating a Patient with RA Using the Oral GnRHAntagonist Elagolix Patient History

-   -   56 year old female with seropositive rheumatoid arthritis with        disease duration of 10 years.    -   Fulfills ACR 2010 classification criteria for rheumatoid        arthritis.

Experimental Treatment

-   -   Is offered experimental GnRH antagonist therapy, elagolix 550 mg        daily for 12 weeks.

Assessment of Response at 4 Months

-   -   Disease activity score (DAS) is reduced with over 0.6 units.    -   A DAS reduction of 0.6 units is defined as a clinical response        to treatment (1).        This example demonstrates how elagolix, a GnRH antagonist, can        improve signs and symptoms in a patient with rheumatoid        arthritis shown with international response criteria.

REFERENCE FOR EXAMPLE 15

-   ¹Van Gestel A M, et al. Development and validation of the European    League Against Rheumatism response criteria for rheumatoid    arthritis. Arthritis Rheum 1996; 39: 34.40.

Example 16 Example of Treating a Patient with RA Using the Oral GnRHAntagonist Relugolix Patient History

-   -   56 year old female with seropositive rheumatoid arthritis with        disease duration of 10 years.    -   Fulfills ACR 2010 classification criteria for rheumatoid        arthritis.

Experimental Treatment

-   -   Is offered experimental GnRH antagonist therapy, relugolix 250        mg daily for 12 weeks.

Assessment of Response at 4 Months

-   -   Disease activity score (DAS) is reduced from 3.5 to 2.5        (remission), or a Good Eular Response    -   A DAS reduction of 0.6 units is defined as a clinical response        to treatment (1).

This example demonstrates that relugolix, a GnRH antagonist, improvessigns and symptoms in a patient with rheumatoid arthritis.

REFERENCE FOR EXAMPLE 16

-   ¹Van Gestel A M, et al. Development and validation of the European    League Against Rheumatism response criteria for rheumatoid    arthritis. Arthritis Rheum 1996; 39: 34.40.

Example 17 Example of Treating a Patient with RA Using the Oral GnRHAntagonist ASP1707

-   -   59 year old female with seropositive rheumatoid arthritis with        disease duration of 8 years.    -   ASP1707 7.5 mg daily for 12 weeks.

Assessment of Response at 4 Months

-   -   Disease activity score (DAS) is reduced with over 0.6 units.    -   A DAS reduction of 0.6 units is defined as a clinical response        to treatment (1).        This example demonstrates how ASP1707, a GnRH antagonist,        improves signs and symptoms in a patient with rheumatoid        arthritis shown with international response criteria.

REFERENCE FOR EXAMPLE 17

-   ¹Van Gestel A M, et al. Development and validation of the European    League Against Rheumatism response criteria for rheumatoid    arthritis. Arthritis Rheum 1996; 39: 34.40.

Example 18 Example of Screening, Diagnosis, and Treatment of Age-RelatedInflammation

A randomized controlled trial is carried out to ascertain whetherinhibiting the effects of GnRH reduces fracture rates, vascular events,myocardial infarctions, and all-cause mortality, with or without gonadalhormone replacement. In a randomized controlled trial approximately 3000men and 3000 women or more, who have moderate peripheral GnRH levels orlevels above 160 pg/mL are allocated to three groups: to either dailyoral 5500 mg elagolix, oral 5500 mg elagolix combined with oestrogen(for example, Activelle 1 mg/0.5 mg women or the amount required totitrate to age/sex pre-chosen levels) or testosterone (for example,Testogel 50 mg every 2-3 days for men or the amount required to titrateto age/sex pre-chosen levels) or placebo. All participants are followedprospectively for the primary trial end point of first-ever myocardialinfarction, stroke, hospitalization for unstable angina, arterialrevascularization, bone fracture, or cardiovascular death. All analysesare performed on an intention-to-treat basis. Additional analysesinclude evaluations of total mortality; the number needed to treat (NNT)to prevent 1 vascular event; whether any observed effect is attributableto LDL reduction, to CRP reduction, or to a combination of bothlipid-lowering and anti-inflammatory effects. After follow up of 5 yearsthere is a significant reduction in the trial primary end point ofvascular events, a reduction in myocardial infarction, and a reductionin all-cause mortality in the elagolix combination with gonadalreplacement group compared to placebo.

This example demonstrates how one could potentially screen forage-related inflammation/systemic inflammation, and treat or prevent itwith a drug that lowers the effects of GnRH.

Example 18A Example of Screening, Diagnosis, and Treatment ofAge-Related Inflammation

A randomized controlled trial is carried out to ascertain whetherinhibiting the effects of GnRH reduces fracture rates, vascular events,myocardial infarctions, and all-cause mortality, with or without gonadalhormone replacement. In a randomized controlled trial approximately 3000men and 3000 women or more, who have moderate peripheral GnRH levels orlevels above 160 pg/mL are allocated to three groups: to either dailyoral 550 mg elagolix, oral 550 mg elagolix combined with oestrogen (forexample, Activelle 1 mg/0.5 mg women or the amount required to titrateto age/sex pre-chosen levels) or testosterone (for example, Testogel 50mg every 2-3 days for men or the amount required to titrate to age/sexpre-chosen levels) or placebo. All participants are followedprospectively for the primary trial end point of first-ever myocardialinfarction, stroke, hospitalization for unstable angina, arterialrevascularization, bone fracture, or cardiovascular death. All analysesare performed on an intention-to-treat basis. Additional analysesinclude evaluations of total mortality; the number needed to treat (NNT)to prevent 1 vascular event; whether any observed effect is attributableto LDL reduction, to CRP reduction, or to a combination of bothlipid-lowering and anti-inflammatory effects. After follow up of 5 yearsthere is a significant reduction in the trial primary end point ofvascular events, a reduction in myocardial infarction, and a reductionin all-cause mortality in the elagolix combination with gonadalreplacement group compared to placebo.This example demonstrates how one could potentially screen forage-related inflammation/systemic inflammation, and treat or prevent itwith a drug that lowers the effects of GnRH.

Example 19 Example of Preventing Age Related Inflammation

An animal study is conducted with 40 fertile female mice agedapproximately 6-20 months in a control group (group 1) receivingplacebo, 40 age and sex matched mice receiving a drug that reduces theactivity of GnRH (group 2), such as cetrorelix 0.5 mg/kg daily, and 40age and sex matched mice receiving the same GnRH inhibitor as group 2,but with the additional replacement of gonadal hormone (oestrogen) toage adjusted pre-chosen levels. All mice are followed prospectively, andevaluated for systemic inflammation markers, atherosclerosis,osteoporosis, LDL, and age at death. The same three interventions andassessments are carried out in three groups of male mice with 40 malemice in each group, aged approximately 6-20 months, replacing thegonadal hormone testosterone to pre-chosen levels in group 3. Micetreated with a drug that reduces the activity of GnRH, such ascetrorelix 0.5 mg/kg daily with or without the replacement of gonadalhormone (preferably with) reach a higher age at death compared with micein the placebo groups. These mice also show less atherosclerosis,osteoporosis and systemic inflammation than mice in the placebo groups.This example demonstrates how a drug to lower the effects of GnRH, suchas cetrorelix can be given to prevent and/or treat age relatedinflammation.

Example 20 Example to Demonstrate Synergistic Effect of GnRH Antagonismwith a Disease-Modifying Drug, Such as Methotrexate, in RheumatoidArthritis Treatment Patient History

-   -   71 year old male with seropositive rheumatoid arthritis with        disease duration of 16 years    -   Fulfilled ACR 2010 classification criteria for rheumatoid        arthritis

Experimental Treatment and Assessment of Response

-   -   Previously failed 3 biologics (etanercept, Actemra, and        rituximab). Last tried rituximab November 2013, but increasing        disease activity and blood tests (CRP and ESR) during the months        thereafter until start of degarelix 16.06.14. Concomitantly used        stable methotrexate 10 mg weekly since 1999.    -   Started degarelix 240 mg induction dose 16.06.14, with 80 mg        every 2 weeks thereafter.    -   Disease activity reduced and he achieved an ACR60 response by        29.09.14 under the introduction of degarelix and stable        concomitant methotrexate and prednisolone.    -   03.10.14 the patient's pulmonary physician stopped methotrexate        treatment due to pleural thickening, and CT imaging supporting        lung fibrosis which was suspected to be due to methotrexate. The        patient had dyspnea which resolved once methotrexate was        stopped.    -   By 21.11.14, the patients disease activity score had increased        from 5.1 (29.9.14) to 5.7 (21.11.14), due to the discontinuation        of methotrexate.        This example demonstrates that the combination of GnRH        antagonist treatment with a DMARD (disease-modifying drug) such        as methotrexate improves signs and symptoms in rheumatoid        arthritis.

Example 21 Background/Purpose

Gonadotropin-releasing hormone (GnRH) agonists lead to increases in thebone formation marker, procollagen type 1 amino-terminal propeptide(P1NP), due to increased bone turnover, followed by decreased bonemineral density (BMD) (1).The effect of GnRH antagonists on bone metabolism is thought to besimilar, as both drugs paradoxically lead to estrogen deprivation.However, data is lacking. We investigated the short-term effects of aGnRH-antagonist, cetrorelix (which rapidly decreases LH and FSH), onlevels of P1NP.

Methods:

In this double-blinded, single-site study in Norway (ClinicalTrials.govNCT00667758), 99 patients with active longstanding RA, were randomisedto predefined intention-to-treat populations using computer-generatedallocation (1:1) in dynamic blocks stratified for sex. Patients wereassigned to subcutaneous cetrorelix (n=48) (5 mg days 1-2, 3 mg days3-5) or placebo (n=51). P1NP was measured on day 10. We investigatedserum P1NP between groups, a predefined secondary endpoint. Detailedefficacy and safety data are presented separately.

Results:

P1NP (m/L) was significantly reduced in the cetrorelix group (−4.21)compared with the placebo group (mean difference−0.57 and −3.45respectively [95% CI:−6.62; −0.29], p=0.033) by day 10; followingsignificant reductions in disease activity markers and TNF-α (logpg/mL). Adverse event rates were similar between groups.

Conclusion:

This study demonstrates antagonizing GnRH with cetrorelix decreasesP1NP, suggesting decreased bone turnover, as well as reductions indisease activity and TNF-α. This is in contrast to observed increases inP1NP with GnRH agonist therapy. Further studies regarding the effect ofupstream hypothalamic-pituitary-gonadal axis hormones on bone metabolismare warranted.

REFERENCE FOR EXAMPLE 20

-   ¹Smith et al. Raloxifene to prevent gonadotropin-releasing hormone    agonist-induced bone loss in men with prostate cancer: A randomized    controlled trial. Journal of Clinical Endocrinology and Metabolism    2004

Example 22 Example of a Synergistic Effect of GnRH Antagonist Treatmentwith Fampridine, a Multiple Sclerosis Drug Patient History

-   -   58 year old female with multiple sclerosis with disease duration        of 8 years.    -   Fulfilled the McDonald 2010 criteria for the diagnosis of        multiple sclerosis (MS).

Experimental Treatment and Assessment of Response

-   -   For the last 2.5 years, she had been taking stable concomitant        fampridine, a potassium channel blocker, for symptomatic        treatment for MS. Her MS disease activity had been increasing        gradually over these 2.5 years, requiring her to use crutches,        and eventually a walker. In December 2014, she started        experimental treatment with degarelix. She received a loading        dose of 240 mg s.c. 08.12.14 and 80 mg s.c. every two weeks        thereafter.    -   By 6 weeks she had responded surprisingly well (refer to        previous example with detailed assessment endpoints).    -   One of the most objective endpoints is the ‘timed 25 meter        walk’.        -   At baseline, she used 16.7 seconds.        -   By 6 weeks she used 15.2 seconds.        -   By 2 months 14.0 seconds.        -   By 4 months 11.9 seconds.        -   She was able to use crutches more frequently and            physiotherapy exercises were completed more easily. She            describes in an interview that for the first time she could            easily complete certain exercises with her under extremities            that she had previously never been able to do during her            physiotherapy visits.    -   Due to this effect, she decided to discontinue her long-term        stable fampridine medication on 05.04.15. This had a detrimental        effect on her timed walk test which increased from 11.9 seconds        to 14.0 seconds on the 29.4.15.    -   In May 2015 we reintroduced fampridine.    -   It is unlikely that the benefits observed were purely due to        fampridine as she had used stable fampridine for 2.5 years        without experiencing such improvements earlier (as confirmed in        her interview as well).        This example demonstrates that the combination of GnRH        antagonist treatment with an MS drug, such as Fampridine,        improved signs and symptoms in multiple sclerosis. This        improvement in progressive MS is likely to also be observed in        the relapsing-remitting type of MS. This is because the        relapsing-remitting type gives similar signs and symptoms as the        progressive type to patients over time.

Example 23 Example of Intra-Articular GnRH-Inhibiting Treatment in aPatient with Osteoarthritis Patient History

-   -   78 year old male with osteoarthritis of both knees diagnosed by        X-ray 10 years ago.    -   Has had synovitis secondary to osteoarthritis in both knees for        the last month, confirmed by synovitis on ultrasound.

Experimental Treatment

-   -   In order to treat the secondary inflammation as a result of his        knee osteoarthritis, he is offered intra-articular GnRH        antagonist treatment, for example cetrorelix 1 mg injected into        both knees. Subcutaneous cetrorelix for injection can be used.

Assessment of Response

-   -   Before and 2 week post injection ultrasound images are taken to        show a decrease in synovitis in the knee joint.        This example demonstrates how a drug that lowers the effects of        GnRH, such as Cetrorelix, may be administered intra-articularly        to treat inflammation.

Example 24 Example of a Patient Using Spiroindoline Derivatives asGonadotropin-Releasing Hormone Receptor Antagonists Patient History

-   -   60 year old female with seropositive rheumatoid arthritis with        disease duration of 20 years.    -   Fulfills ACR 2010 classification criteria for rheumatoid        arthritis.

Experimental Treatment

-   -   Treated with a spiroindoline derivative as a GnRH antagonist        orally 200 mg daily for 12 weeks.

Assessment of Response at 4 Months

-   -   Disease activity score (DAS) is reduced with over 0.6 units.    -   A DAS reduction of 0.6 units is defined as a clinical response        to treatment (1).        This example demonstrates how spiroindoline derivatives as GnRH        antagonists, can improve signs and symptoms in a patient with        rheumatoid arthritis shown with international response criteria.

REFERENCE FOR EXAMPLE 24

-   ¹Van Gestel A M, et al. Development and validation of the European    League Against Rheumatism response criteria for rheumatoid    arthritis. Arthritis Rheum 1996; 39: 34.40.

Example 25 Example of a Patient with Tendonitis Treated with a GnRHAntagonist Injection Patient History

-   -   61 year old male diagnosed with tendonitis of the elbow with        ultrasound confirming tendonitis.

Experimental Treatment

-   -   Treated with an injection of GnRH antagonist, ganirelix 1.5 mg,        around the affected tendon.

Assessment of Response

-   -   Before and 2 week post injection ultrasound images are taken to        show a decrease in tendonitis of the elbow.        This example demonstrates how a drug that lowers the effects of        GnRH, such as ganirelix, may be administered around tendons to        treat inflammation. Such injections may be administered over the        long-term, for example every 3 months to maintain optimal        effect.

Example 26 Example of a Patient with Psoriatic Arthritis Treated withthe Oral GnRH Antagonist ASP1707 Patient History

-   -   48 year old male with psoriatic arthritis    -   Fulfills CASPAR 2006 classification criteria for psoriatic        arthritis.

Experimental Treatment

-   -   Is offered experimental GnRH antagonist therapy, ASP1707 3 mg        daily for 12 weeks.

Assessment of Response at 4 Months

-   -   Disease activity score (DAS) is reduced with over 0.6 units.    -   A DAS reduction of 0.6 units can be defined as a clinical        response to treatment (1).        This example demonstrates how ASP1707, a GnRH antagonist,        improves signs and symptoms in a patient with rheumatoid        arthritis shown with international response criteria.

REFERENCE FOR EXAMPLE 26

-   ¹Mease P J, et al. Psoriatic arthritis assessment tools in clinical    trials. Ann Rheum Dis 2005; (Suppl II):ii49-ii54. doi:    10.1136/ard.2004.034165.

Example 27

We analysed peripheral GnRH levels in patients with various autoimmunediseases using ELISA. We found that patients with autoimmune diseaseshad higher levels of peripheral GnRH than healthy controls. We alsofound that GnRH antagonist treatment over a period of months reducedperipheral GnRH levels to some extent in patients with variousautoimmune diseases. Due to the nature of ELISA assays, theconcentration in pg/mL should not necessarily be regarded as absolute,and may be interpreted as relative.

RA Patient A Baseline GnRH 394 pg/mL, after 3 months of degarelix withloading dose 240 mg, and maintenance doses 80 mg every 2 weeks, GnRH 359pg/mL. During this period CRP was reduced from 142 mg/L to 54 mg/L anddisease activity reduced significantly from 8.6 to 4.2 in DAS score.Multiple Sclerosis Patient B Baseline GnRH 318 pg/mL, reduced to 210pg/mL after 1 month of GnRH antagonist treatment; degarelix 240 mgloading dose and 80 mg every 2 weeks for maintenance. During thatmonth/6 weeks her general function had improved, for example MSIS-29 hadreduced from 121 to 110. Psoriasis Patient C Baseline GnRH 300 pg/mLreduced to 274 pg/mL after 3 months degarelix treatment with loadingdose 240 mg, and maintenance doses of 80 mg every 2 weeks. During thatperiod his PASI score reduced by more than 20%. Healthy control 1baseline GnRH 162 pg/mL, healthy control 2 baseline GnRH 120 pg/mL,healthy control 3 baseline GnRH 153 pg/mL.

This example shows that patients with inflammatory diseases may havehigher peripheral GnRH levels than healthy controls, and this exampleshows how assessing peripheral levels of GnRH can be used to generallyused to diagnose inflammatory diseases and evaluate treatmenteffect/prognostic evaluation.

Example 28 A Fusion GnRH Antagonist Compound

Functionalization of the PEG polymer at one or both terminal is carriedout. The chemically active or activated derivatives of the PEG polymerare prepared to attach the PEG to the desired molecule, the GnRHantagonist or drug to lower GnRH activity. The overall PEGylationprocess is either a solution phase batch process or an on-columnfed-batch process. During the simple and commonly adopted batch processreagents are mixed together in a suitable buffer solution, preferably ata temperature between 4 and 6° C. The choice of the suitable functionalgroup for the PEG derivative is based on the type of available reactivegroup on the molecule that is coupled to the PEG. For proteins, typicalreactive amino acids include lysine, cysteine, histidine, arginine,aspartic acid, glutamic acid, serine, threonine, tyrosine. TheN-terminal amino group and the C-terminal carboxylic acid can also beused as a site specific site by conjugation with aldehyde functionalpolymers. For first generation PEG derivatives the PEG polymer reactswith a group that is reactive with hydroxyl groups, typicallyanhydrides, acid chlorides, chloroformates and carbonates. For secondgeneration PEG derivatives, more efficient functional groups such asaldehyde, esters, amides etc. is made available for conjugation.Heterobifunctional PEGs are very useful in linking two entities, where ahydrophilic, flexible and biocompatible spacer is needed. Preferred endgroups for heterobifunctional PEGs are maleimide, vinyl sulfones,pyridyl disulfide, amine, carboxylic acids and NHS esters. Thirdgeneration pegylation agents, where the shape of the polymer has beenbranched, Y-shaped or comb-shaped are available which show reducedviscosity and lack of organ accumulation. These compounds are tested inanimal studies, showing that they do not cross the blood brain barrierdue to their physically large size. Some of these compounds can haveextended half-lives. They do not act upon the pituitary GnRH receptors.They are shown to only act upon peripheral GnRH receptors, avoiding sideeffects of decreased oestrogen and testosterone in females and malesrespectively. They are administered through several routes, includingoral routes. This enables long-term administration for years, and insome examples maintenance of the menstrual cycle in premenopausal women.

Example 29 A Monoclonal Antibody Compound

Mice are immunized to stimulate anti-GnRH antibody production. Antibodyforming cells are isolated from the spleen. Tumor cells are grown intissue culture. Antibody-forming cells that are isolated from the spleenare fused with cultivated tumor cells to form hybridomas. Hybridomas arescreened for antibody production. Antibody-producing hybridomas arecloned. Monoclonal antibodies are isolated for cultivation. Themonoclonal antibody works by binding with high affinity to peripheralGnRH, preventing the binding of GnRH to its receptor. The antibody is alarge molecule that is unable to pass the BBB therefore central GnRHreceptors remain unaffected, thereby the reproductive hypothalamicpituitary gonadal axis is unaffected.

Example 30 Example Treatment Using the Oral GnRH Antagonist RelugolixPatient History

-   -   38 year old male with spondyloarthritis with disease duration of        5 years.

Experimental Treatment

-   -   Is offered experimental GnRH antagonist therapy, relugolix 400        mg daily for 12 weeks.

Assessment of Response at 4 Months

-   -   Disease activity in ankylosing spondylitis/spondyloarthritis is        measured by the BASDAI score. His BASDAI is 5 at baseline. It is        1.2, in remission, at 12 weeks.    -   This example demonstrates that relugolix, a GnRH antagonist,        improves signs and symptoms in a patient with spondyloarthritis.

Example 31 Example Treatment Using the Oral GnRH Antagonist ElagolixPatient History

-   -   38 year old female with systemic sclerosis (scleroderma) with        disease duration of 1 year.

Experimental Treatment

-   -   Is offered experimental GnRH antagonist therapy, elagolix 600 mg        daily for 12 weeks.

Assessment of Response

-   -   Digital ulcers decrease from 6 to 2    -   Health assessment questionnaire improves from 2.0 to 1.0    -   This example demonstrates that elagolix, a GnRH antagonist,        improves signs and symptoms in a patient with systemic sclerosis        (scleroderma).

Example 32 Example Treatment Using the Oral GnRH Antagonist ASP1707 toReduce Cancer-Related Inflammation

Inflammation and cancer are linked inextricably. Inflammation has beendescribed as the 7^(th) hallmark of cancer. The majority ofcancer-related symptoms are associated with inflammation.

Patient History

-   -   68 year old male with colon cancer with disease duration of 1        year. Suffers from signs and symptoms of cancer-related        inflammation, such as pain, anorexia, and fatigue.

Experimental Treatment

-   -   Is offered experimental treatment, ASP1707 8 mg daily, to reduce        signs and symptoms associated with cancer related inflammation

Assessment of Response

-   -   At 3 months patient experiences reduced signs and symptoms of        cancer related inflammation, such as pain, anorexia and fatigue.    -   At 3 months, his high baseline CRP of 40 mg/L is decreased to 12        mg/L.    -   This example demonstrates that ASP1707, a GnRH antagonist,        improves signs and symptoms in a patient with cancer-related        inflammation.

Example 33 Example of a GnRH Antagonist Treatment for Colitis PatientHistory

-   -   55 year old female with ulcerative colitis for the past 10 years

Experimental Treatment

-   -   Is offered ganirelix 2 mg daily for 6 months

Assessment of Response

-   -   By 6 months she experiences clinical remission with a partial        Mayo score reduction from 3 to O.        This example demonstrates how a GnRH antagonist may be given to        treat an inflammatory bowel disease.

Example 34

The association of GnRH and TNFα in the periphery of patients withrheumatoid arthritis was assessed and the results are shown in FIG. 14.All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled inmolecular biology, in vitro fertilization, development, or relatedfields are intended to be within the scope of the following claims.

Example 35 Treatment Regime for a Patient Who is a Post-MenopausalFemale with Rheumatoid Arthritis with the Oral GnRH Antagonist ASP1707Key Patient Inclusion Criteria:

-   -   Subject is a natural post-menopausal female whose last regular        menstrual cycle is at least 24 months ago at the time of        informed consent.    -   Follicle-stimulating hormone (FSH)≧30 IU/mL at screening.    -   Subject has RA that was diagnosed according to the 1987 ACR        criteria or the 2010 ACR/EULAR criteria for at least 6 months.    -   Subject meets the ACR 1991 Revised Criteria for the        Classification of Global Functional Status in RA Class I, II or,        III at screening.    -   At screening subject has active RA as evidenced by both of the        following:        -   ≧6 tender/painful joints (using 68-joint assessment)        -   ≧6 swollen joints (using 66-joint assessment).    -   CRP of >0.3 mg/dL or ESR of >28 mm/hr at screening.    -   Subject who continuously received methotrexate (MTX) for at        least 90 days prior to the start of screening and who is able to        continue stable dose of MTX from at least 28 days prior to the        start of screening through the end of treatment, or follow-up        period.

Experimental Treatment:

-   -   Patient is offered experimental GnRH antagonist therapy, ASP1707        30 mg twice daily for 12 weeks orally. Primary Assessment is        ACR20 response rate at 12 weeks. Other assessments include ACR20        response rate at 1, 2, 4 and 8 weeks, and the following        variables at 1, 2, 4, 8 and 12 weeks:        -   ACR50 response rate.        -   ACR70 response rate.        -   Change from baseline in DAS28-CRP and DAS28-ESR score        -   Change from baseline in Tender Joint Count (68 joints).        -   Change from baseline in Swollen Joint Count (66 joints).        -   Percentage of subjects achieving DAS28-CRP and DAS28-ESR            score for remission (<2.6)        -   Percentage of subjects achieving DAS28-CRP score and            DAS28-ESR score for low disease activity (<3.2)        -   Change from baseline in CRP and ESR        -   Percentage of subjects achieving EULAR response criterion of            “Good Response”.        -   Percentage of subjects achieving EULAR response criterion of            “Good Response” or “Moderate Response”.        -   Percentage of subjects achieving ACR/EULAR score for            remission.        -   Percentage of subjects achieving SDAI score<3.3 (SDAI            remission)        -   Change from baseline in the SDAI score        -   Change from baseline for the HAQ-DI    -   ASP1707 30 mg b.i.d. is expected to inhibit LH level by 90%.        Other doses of ASP1707 to be administered are presented in the        table below.

Dose (mg) Administration 5 twice daily 10 twice daily 15 twice daily 20twice daily 45 twice daily 50 twice daily

-   -   Assuming placebo+MTX response rate for ACR20 at Week 12 is 30%,        ASP1707+MTX response rate for ACR20 at Week 12 is expected to be        60%.        This example is expected to demonstrate how ASP 1707, a GnRH        antagonist, improves signs and symptoms in a patient with        rheumatoid arthritis shown with international response criteria.

1. A method of treating or preventing an inflammatory condition in asubject, wherein said inflammatory condition is selected from aninflammatory disease, chronic inflammation, age-related inflammation orinflammatory peripheral GnRH, said method comprising: administeringASP1707 or a pharmaceutically acceptable salt thereof to said subject,wherein ASP1707 or the pharmaceutically acceptable salt thereof isadministered long-term to said subject for a period of at least 12weeks.
 2. The method of claim 1, wherein said inflammatory condition isan autoimmune disease.
 3. The method of claim 1, wherein saidinflammatory disease is rheumatoid arthritis, an inflammatory boweldisease, a spondyloarthritis, systemic sclerosis (scleroderma),psoriasis, nephritis, multiple sclerosis or osteoarthritis.
 4. Themethod of claim 3, wherein said disease is rheumatoid arthritis.
 5. Themethod of claim 1, wherein said inflammatory disease is ankylosingspondylitis.
 6. The method of claim 3, wherein said inflammatory boweldisease is colitis or Crohn's disease.
 7. The method of claim 1, whereinASP1707 or the pharmaceutically acceptable salt thereof is for use intreating or preventing osteoporosis or for increasing bone mineraldensity.
 8. The method of claim 1, wherein ASP1707 or thepharmaceutically acceptable salt thereof is for use in treating acardiovascular disease or metabolic syndrome, or for decreasing the riskof a cardiovascular event or of developing coronary heart disease ormetabolic syndrome by treating one or more risk factors forcardiovascular disease in a subject.
 9. The method of claim 8, whereinASP1707 or the pharmaceutically acceptable salt thereof decreases HBA1c,decreases blood pressure, or increases HDL levels in said subject. 10.The method of claim 8, wherein ASP1707 or the pharmaceuticallyacceptable salt thereof is for use in decreasing blood pressure.
 11. Themethod of claim 1, wherein the inflammatory condition is systemicchronic inflammation.
 12. The method of claim 11, wherein said systemicchronic inflammation is age-related systemic chronic inflammation. 13.The method of claim 11, wherein the systemic chronic inflammation islow-level inflammation.
 14. The method of claim 1, wherein theinflammatory condition is cancer inflammation.
 15. The method of claim1, wherein said method is for the treatment or prevention of low levelsystemic chronic inflammation in a subject who is without overt clinicalsymptoms of inflammatory disease.
 16. The method of claim 1, whereinsaid method is for the treatment or prevention of peripheralinflammatory GnRH in a subject who exhibits a level of peripheral GnRHwhich is 160 pg/ml or above.
 17. The method of claim 16, wherein thesubject is healthy or is without overt clinical symptoms of inflammatorydisease,
 18. The method of claim 1, wherein ASP1707 or thepharmaceutically acceptable salt thereof is in the form of a conjugatewith a polymer which serves to inhibit passage of ASP1707 or the saltthereof across the blood brain barrier.
 19. The method of claim 18,wherein the polymer is a polypeptide, a polyethylene glycol (PEG) or apolysaccharide.
 20. The method of claim 1, wherein ASP1707 or thepharmaceutically acceptable salt thereof is used in combination with oneor more additional active agents.
 21. The method of claim 20, whereinthe additional active agent is an agent useful for the treatment ofinflammation, particularly an agent useful in the treatment of aninflammatory disease or an autoimmune disease, including rheumatoidarthritis, an inflammatory bowel disease such as colitis or Crohn'sdisease, a spondyloarthritis, systemic sclerosis (scleroderma),psoriasis, nephritis, multiple sclerosis, osteoarthritis and ankylosingspondylitis.
 22. The method of claim 20, wherein ASP1707 or thepharmaceutically acceptable salt thereof and the additional active agenthave a synergistic effect.
 23. The method of claim 20, wherein ASP1707or the pharmaceutically acceptable salt thereof is used in combinationwith an additional active agent which is a sex hormone, includingoestrogen or testosterone, or an agent useful in sex hormonesubstitution therapy, including LH or FSH.
 24. The method of claim 23,wherein the sex hormone is titrated to a desired or selected level. 25.The method of claim 20, wherein the additional active agent is selectedfrom an anti-rheumatic agent, a non-steroidal anti-inflammatory drug(NSAID), a biologic agent, an analgesic, a steroid, a glucocorticoid, anagent used to treat osteoporosis and an agent used to treat multiplesclerosis.
 26. The method of claim 20, wherein said one or moreadditional active agents are selected from the group consisting ofmethotrexate, fampridine, daivobet, oestrogen and testosterone.
 27. Themethod of claim 1, wherein ASP1707 or the pharmaceutically acceptablesalt thereof is administered to said subject for at least five months.28. The method of claim 27, wherein ASP1707 or the pharmaceuticallyacceptable salt thereof is administered to said subject for at least oneyear.
 29. The method of claim 1, wherein ASP1707 or the pharmaceuticallyacceptable salt thereof is administered to said subject multiple times.30. The method of claim 29, wherein ASP1707 or the pharmaceuticallyacceptable salt thereof is administered to said subject multiple timesper day, daily, weekly, or monthly.
 31. The method of claim 30, whereinASP1707 or the pharmaceutically acceptable salt thereof is administeredto said subject with a single loading dose followed by a lowermaintenance dose administered multiple times per day, daily, weekly, ormonthly.
 32. The method of claim 31, wherein ASP1707 or thepharmaceutically acceptable salt thereof is in the form of a sustainedrelease preparation and is for administration at an initial loading doseof 20 to 1000 mg, e.g. 240 mg, followed by a maintenance dose of either(i) 60 to 1000 mg, e.g. 80-160 mg, every 2 weeks, or (ii) 30 to 300 mg,e.g. 40-150 mg, every week.
 33. The method of claim 1, wherein ASP1707or the pharmaceutically acceptable salt thereof is administered to saidsubject at a dosage of 0.5 to 200 mg/day, administered 1 or more times aday.
 34. The method of claim 33, wherein ASP1707 or the pharmaceuticallyacceptable salt thereof is administered to said subject at a dosage of10 to 100 mg/day, administered 1 or more times a day, preferably twice aday.
 35. The method of claim 34, wherein ASP1707 or the pharmaceuticallyacceptable salt thereof is administered to said subject at a dosage of50 to 70 mg/day, preferably 60 mg/day, administered 1 or more times aday, preferably twice a day.
 36. The method of claim 1, wherein ASP1707or the pharmaceutically acceptable salt thereof is administered to saidsubject 2, 3, 4, 5 or 6 times a day.
 37. The method of claim 34, whereinASP1707 or the pharmaceutically acceptable salt thereof is administeredto said subject twice a day, each dose being in the range from 5 to 50mg, preferably 30 mg.
 38. The method of claim 1, wherein the level ofperipheral GnRH in said subject is determined prior to administration ofASP1707 or the pharmaceutically acceptable salt thereof, or is monitoredover a period of time, prior to and/or during administration of ASP1707or the pharmaceutically acceptable salt thereof.
 39. The method of claim38, wherein ASP1707 or the pharmaceutically acceptable salt thereof isadministered to said subject if the level of peripheral GnRH is 160pg/ml or above.
 40. The method of claim 1 wherein said subject is apost-menopausal female.
 41. The method of claim 1 wherein ASP1707 or thepharmaceutically acceptable salt thereof is administered orally to saidsubject.
 42. The method of claim 1 wherein said subject is already beingtreated for the said inflammatory condition, preferably withmethotrexate.
 43. The method of claim 1, wherein said inflammatorycondition is rheumatoid arthritis, said subject is a post-menopausalfemale, and ASP1707 or the pharmaceutically acceptable salt thereof isadministered orally to said subject twice daily, with a total daily dosein the range from 10 to 100 mg, preferably 60 mg, each of the two dailydoses being of equal amounts in the range from 5 to 50 mg, preferably 30mg.
 44. A method of treating or preventing an inflammatory condition ina subject, selected from an inflammatory disease, chronic inflammation,age-related inflammation or inflammatory peripheral GnRH, preferably anautoimmune disease, said method comprising: administering a combinationof ASP1707 or a pharmaceutically acceptable salt thereof and anadditional active agent for treatment of said condition to said subject,wherein ASP1707 or the pharmaceutically acceptable salt thereof and saidadditional active agent are administered long-term to said subject for aperiod of at least 12 weeks.
 45. The method of claim 40, wherein saidadditional active agent is an agent useful for the treatment ofinflammation, particularly an agent useful in the treatment of aninflammatory disease or an autoimmune disease, including rheumatoidarthritis, an inflammatory bowel disease such as colitis or Crohn'sdisease, a spondyloarthritis, systemic sclerosis (scleroderma),psoriasis, nephritis, multiple sclerosis, osteoarthritis and ankylosingspondylitis, preferably wherein the additional active agent is selectedfrom an anti-rheumatic agent, a non-steroidal anti-inflammatory drug(NSAID), a biologic agent, an analgesic, a steroid, a glucocorticoid, anagent used to treat osteoporosis and an agent used to treat multiplesclerosis, e.g. wherein said additional active agent is selected fromthe group consisting of methotrexate, fampridine, daivobet, oestrogenand testosterone.
 46. The method of claim 40, wherein the ASP1707 or thepharmaceutically acceptable salt thereof is in the form of a conjugatewith a polymer which serves to inhibit passage of ASP1707 or the saltthereof across the blood brain barrier, and/or wherein ASP1707 or thepharmaceutically acceptable salt thereof is administered to said subjectfor at least five months, is administered to said subject multipletimes, is administered to said subject at a dosage of 0.5 to 200 mg/day1 or more times a day, or is administered to said subject 2, 3, 4, 5 or6 times a day.
 47. A conjugate comprising ASP1707 or a pharmaceuticallyacceptable salt thereof linked to a polymer which serves to inhibitpassage of ASP1707 or the pharmaceutically acceptable salt thereofacross the blood brain barrier.
 48. A pharmaceutical compositioncomprising a conjugate as defined in claim 43, together with at leastone pharmaceutically acceptable carrier or excipient.